Plant transactivation interaction motifs and uses thereof

ABSTRACT

This disclosure concerns compositions and methods for increasing the expression of a polynucleotide of interest. Some embodiments concern novel transactivation polypeptides and variants thereof that have been identified in plants, and methods of using the same. Particular embodiments concern the use of at least one DNA-binding polypeptide in a fusion protein to target at least one transactivation polypeptide or variant thereof to a specific binding site on a nucleic acid comprising the polynucleotide of interest, such that its expression may be increased.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/594,245, filed Feb. 2, 2012, the disclosure ofwhich is hereby incorporated herein in its entirety by this reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to plant biotechnology. Embodimentsrelate to polypeptides (e.g., a fusion protein) comprising a novel orsynthetic transcription factor interaction motif from a planttransactivator. Some embodiments relate to the use of such a protein toexpress a nucleic acid of interest or to increase the expression of anucleic acid of interest. Some embodiments relate to polynucleotidesencoding a protein comprising a novel or synthetic transcription factorinteraction motif from a plant transactivator. Particular examplesrelate to host cells, tissues, and/or organisms comprising a polypeptideor polynucleotide of the invention.

BACKGROUND

The introduction of cloned and isolated genes into plant cells (genetictransformation), and the subsequent regeneration of transgenic plants,is widely used to make genetic modifications of plants and plantmaterials. Genetic transformation of plants to introduce a desirabletrait (e.g., improved nutritional quality; increased yield; pest ordisease resistance; stress tolerance; and herbicide resistance) is nowcommonly used to produce new and improved transgenic plants that expressthe desirable trait. DNA is typically randomly introduced into thenuclear or plastid DNA of a eukaryotic plant cell, and cells containingthe DNA integrated into the cell's DNA are then isolated and used toproduce stably-transformed plant cells. Often, it is desirable togenetically engineer a single plant variety to express more than oneintroduced trait by introducing multiple coding sequences, which maycomprise similar (or identical) regulatory elements.

The expression of transgenes (as well as endogenous genes) is controlledthrough mechanisms involving multiple protein-DNA and protein-proteininteractions. Through such interactions, nucleic acid regulatoryelements (e.g., promoters and enhancers) can impart patterns ofexpression to a coding sequence that are either constitutive orspecific. For example, a promoter may lead to increased transcription ofa coding sequence in specific tissues, during specific developmentperiods, or in response to environmental stimuli. Unfortunately, theinherent attributes of conventional promoters for transgene expressionlimit the range of expression control that they may be used to exert ina host cell. One practical limitation of conventional promoters is thatit is difficult to finely tune the expression level of an introducedgene due to limitations in promoter strength and to the silencing oftransgene expression by particularly strong promoters or thesimultaneous use in the same cell of many copies of the same promoter.It can also be desirable to initiate or increase expression ofendogenous or native genes.

Transactivators are proteins that function by recruiting throughprotein-protein interactions a number of different proteins involved inDNA transcription (e.g., nucleosome-remodeling complexes; the mediatorcomplex; and general transcription factors, such as TFIIB, TBP, andTFIIH) to initiate or enhance the rate of transcription by affectingnucleosome assembly/disassembly, pre-initiation complex formation,promoter clearance, and/or the rate of elongation. The protein-proteininteractions of transactivators and their binding partners involvediscrete internal structural elements within the transactivators knownas “transactivation domains (TADs).” TADs are thought to share littleprimary sequence homology and adopt a defined structure only uponbinding to a target. Sigler (1988) Nature 333:210-2. Though acidic andhydrophobic residues within the TADs are thought to be important (see,e.g., Cress and Triezenberg (1991) Science 251(4989):87-90), thecontribution of individual residues to activity is thought to be small.Hall and Struhl (2002) J. Biol. Chem. 277:46043-50.

The Herpes Simplex virion protein 16 (VP16) is a transactivator thatfunctions to stimulate transcription of viral immediate early genes inHSV-infected cells. As with other transactivators, VP16 activatestranscription through a series of protein-protein interactions involvingits TAD, which is highly acidic. The acidic TAD of VP16 has been shownto interact with several partner proteins both in vitro and in vivo. Forexample, the TAD of VP 16 contains an interaction motif that interactsdirectly with the Tfb1 subunit of TFIIH (Langlois et al. (2008) J. Am.Chem. Soc. 130:10596-604), and this interaction is correlated with theability of VP16 to activate both the initiation and elongation phase oftranscription for viral immediate early genes.

BRIEF SUMMARY OF THE DISCLOSURE

Described herein are novel TAD protein-protein interaction motifs thathave been isolated from plant transactivator proteins, and nucleic acidsencoding the same. These novel interaction motifs may be utilized in asynthetic TAD to confer gene regulatory properties upon a polypeptidecomprising the TAD. For example, some embodiments include atranscriptional activator fusion protein that comprises a DNA-bindingdomain polypeptide and such a TAD polypeptide. Depending upon theparticular DNA-binding domain that is fused to the TAD in thetranscriptional activator fusion protein, transactivation may be used toincrease the expression of a gene of interest. For example, aheterologous polynucleotide to which the DNA-binding domain binds may beoperably linked to the gene of interest, thereby targeting the fusionprotein (and its functional TAD), the binding of which will increase theexpression of the gene of interest. Alternatively, a DNA-binding domainmay be engineered to bind an endogenous polynucleotide that is operablylinked to, or proximal to, the gene of interest. Upon binding of atranscriptional activator fusion protein to a target DNA binding site,transcription of a gene operably linked to the target DNA binding sitemay be stimulated.

Also described herein are synthetic variant TAD protein-proteininteraction motifs, and nucleic acids encoding the same. In someexamples, a synthetic variant TAD protein-protein interaction motif isengineered by introducing one or more mutations (e.g., a conservativemutation, or a mutation identified in an ortholog of the interactionmotif) into the TAD of a transactivator (e.g., a plant transactivator).Surprisingly, a synthetic variant TAD generated in this manner thatcomprises a variant interaction motif may confer gene regulatoryproperties different from the unmodified TAD when coupled to aDNA-binding domain in a transcriptional activator fusion protein. Forexample, particular synthetic variant TADs that comprise a variantinteraction motif may enhance the level of transcriptional activationconferred by the naturally-occurring TAD interaction motif whenexpressed in the same position in a fusion protein comprising aDNA-binding domain.

Some embodiments include a synthetic transcriptional activator fusionprotein. In particular embodiments, the fusion protein may increasetranscription of a gene of interest, wherein the fusion proteincomprises a first polypeptide comprising a DNA-binding domainoperatively linked to a second polypeptide comprising a TAD interactionmotif. In some examples, the TAD interaction motif may be selected fromthe group of TAD interaction motifs consisting of SEQ ID NOs: 10-16. Forexample and without limitation, the TAD interaction motif may becomprised within a TAD comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2-8 and SEQ ID NOs: 100-106. In someexamples, the TAD interaction motif may be a variant TAD interactionmotif having, for example and without limitation, an amino acid sequenceselected from the group consisting of SEQ ID NOs: 17-58. For example andwithout limitation, such a variant TAD interaction motif may becomprised within a TAD comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 107-120.

Some embodiments include a polynucleotide that encodes a synthetictranscriptional activator fusion protein comprising a first polypeptidecomprising a DNA-binding domain operatively linked to a secondpolypeptide comprising a TAD interaction motif. A DNA-binding domainpolypeptide may be any DNA-binding domain that binds specifically to aparticular target DNA binding site. For example and without limitation,the DNA-binding domain polypeptide may be a polypeptide selected fromthe group consisting of a zinc finger DNA-binding domain; UPADNA-binding domain; GAL4; TAL; LexA; a Tet repressor; LacR; and asteroid hormone receptor. In particular examples, a DNA-bindingdomain-encoding sequence may be selected from the group consisting ofSEQ ID NO: 67; SEQ ID NO: 68; and SEQ ID NO: 99. In particular examples,the polynucleotide may comprise a DNA-binding protein-encoding sequencethat is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to asequence selected from the group consisting of SEQ ID NO: 67; SEQ ID NO:68; and SEQ ID NO: 99.

In some examples, the polynucleotide may comprise a TAD interactionmotif-encoding sequence that encodes a TAD interaction motif or variantTAD interaction motif, e.g., having a sequence selected from SEQ ID NOs:10-58. Particular embodiments include a polynucleotide that encodes atranscriptional activator fusion protein comprising at least one TADinteraction motif. Particular embodiments include a polynucleotide thatencodes a transcriptional activator fusion protein comprising at leastone DNA-binding domain.

Examples of polynucleotides that encode a synthetic transcriptionalactivator fusion protein according to some embodiments of the inventioninclude polynucleotides comprising at least one nucleotide sequenceencoding a DNA-binding domain and at least one nucleotide sequenceencoding a TAD interaction motif (or variant thereof), wherein thepolynucleotide comprises, for example and without limitation, at leastone nucleotide sequence selected from the group consisting of: SEQ IDNOs: 79-93; a nucleotide sequence that is substantially identical to oneof SEQ ID NOs: 79-93; a nucleotide sequence having at least 80% sequenceidentity to one of SEQ ID NOs: 79-93; a nucleotide sequence having atleast 85% sequence identity to one of SEQ ID NOs: 79-93; a nucleotidesequence having at least 90% sequence identity to one of SEQ ID NOs:79-93; a nucleotide sequence having at least 95% sequence identity toone of SEQ ID NOs: 79-93; a nucleotide sequence having at least 97%sequence identity to one of SEQ ID NOs: 79-93; a nucleotide sequencehaving at least 98% sequence identity to one of SEQ ID NOs: 79-93; anucleotide sequence having at least 99% sequence identity to one of SEQID NOs: 79-93; the complement of a polynucleotide that is specificallyhybridizable to at least one of SEQ ID NOs: 79-93; and the reversecomplement of a polynucleotide that is specifically hybridizable to atleast one of SEQ ID NOs: 79-93.

In some embodiments, a polynucleotide that encodes a transcriptionalactivator fusion protein may be incorporated into a recombinant vector,for example, to provide expression of the protein in a host cell.Accordingly, some examples include a vector comprising at least onepolynucleotide of the invention, and/or a host cell into which such avector has been introduced.

Also described herein are means for transactivation of plant geneexpression. As used herein, a “means for transactivation of plant geneexpression” includes a polypeptide selected from the group consisting ofSEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO:22; SEQ ID NO: 28; SEQ ID NO: 46; and SEQ ID NO: 52. In someembodiments, a synthetic protein comprising at least one means fortransactivation of plant gene expression may be used to modulate theexpression of a gene of interest in a plant cell.

Additionally, described herein are means for increasing gene expressionthat are derived from ERF2. As used herein, a “means for increasing geneexpression that is derived from ERF2” includes a polypeptide selectedfrom the group consisting of SEQ ID NOs: 17-22 and SEQ ID NO: 121.Additionally described are means for increasing gene expression that arederived from PTI4. As used herein, a “means for increasing geneexpression that is derived from PTI4” includes a polypeptide selectedfrom the group consisting of SEQ ID NOs: 23-28 and SEQ ID NO: 122.Additionally described are means for increasing gene expression that arederived from AtERF1. As used herein, a “means for increasing geneexpression that is derived from AtERF1” includes a polypeptide selectedfrom the group consisting of SEQ ID NOs: 29-34 and SEQ ID NO: 123.Additionally described are means for increasing gene expression that arederived from ORCA2. As used herein, a “means for increasing geneexpression that is derived from ORCA2” includes a polypeptide selectedfrom the group consisting of SEQ ID NOs: 35-40 and SEQ ID NO: 124.Additionally described are means for increasing gene expression that arederived from DREB1A. As used herein, a “means for increasing geneexpression that is derived from DREB1A” includes a polypeptide selectedfrom the group consisting of SEQ ID NOs: 41-46 and SEQ ID NO: 125.Additionally described are means for increasing gene expression that arederived from CBF1. As used herein, a “means for increasing geneexpression that is derived from CBF1” includes a polypeptide selectedfrom the group consisting of SEQ ID NOs: 47-52 and SEQ ID NO: 126.Additionally described are means for increasing gene expression that arederived from DOF1. As used herein, a “means for increasing geneexpression that is derived from DOF1” includes a polypeptide selectedfrom the group consisting of SEQ ID NOs: 53-58 and SEQ ID NO: 127.

Also described herein are methods for increasing gene expressionutilizing a synthetic transcriptional activator fusion protein. Inexamples, an expression vector comprising a polynucleotide encoding asynthetic transcriptional activator fusion protein may be introducedinto a host cell (e.g., a plant cell, yeast cell, mammalian cell, andimmortalized cell) comprising a gene of interest operably linked to atarget DNA binding site for the fusion protein. Expression of the fusionprotein in the host cell, and subsequent binding of the fusion proteinto the operably linked target DNA binding site, may result intranscription initiation or increased transcription of the gene ofinterest. In particular examples, the target DNA binding site may beintroduced into the host cell, such that the target DNA binding site isoperably linked to the gene of interest. In further examples, asynthetic transcriptional activator fusion protein may comprise aDNA-binding domain polypeptide that is engineered to bind to a targetDNA binding site that is operably linked to the gene of interest.

In some embodiments, a vector comprising a polynucleotide encoding asynthetic transcriptional activator fusion protein may be introducedinto a host cell, such that the polynucleotide is subsequentlyintegrated into the genomic DNA of the host cell (e.g., via homologousrecombination). Thus, a synthetic transcriptional activator fusionprotein, and moreover a nucleic acid encoding the same, may be comprisedwithin a transgenic organism (e.g., a transgenic plant). Accordingly,such transgenic organisms are also described herein. In examples, anucleic acid encoding a synthetic transcriptional activator fusionprotein may be either integrated randomly, or at a predeterminedlocation, in the genome of a cell in the transgenic organism.

Further described are methods for expressing a gene of interestutilizing a synthetic transcriptional activator fusion protein and/or anucleic acid encoding the same. In some embodiments, a vector comprisinga polynucleotide encoding a synthetic transcriptional activator fusionprotein may be introduced into a host cell comprising a gene of interestoperably linked to a target DNA binding site for the fusion protein. Insome examples, the synthetic transcriptional activator fusion proteincomprises a means for transactivation of plant gene expression. Afterthe vector is introduced into the host cell, expression of the gene ofinterest may be initiated or increased, thereby producing the expressionproduct of the gene of interest in the host cell, for example, in anamount according to the regulatory control of the fusion protein. Suchexpression products may be isolated and/or purified from the host cellaccording to any method known in the art.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 includes an identified interaction motif of the VP 16transactivation domain (TAD), subdomain II (SEQ ID NO: 9). The asterisksindicate the amino acids of VP16 transactivation domain, subdomain II,which are proposed to directly contact the Tfb1 subunit of TFIIH asproposed by Langlois et al. (2008) J. Am. Chem. Soc. 130:10596-604.

FIG. 2 includes an alignment of the VP16 transactivation subdomain IIwith the identified plant TADs. The listed plant TADs contain aninteraction motif. The aligned interaction motifs are highlighted. Theresidues of the interaction motif of subdomain II from VP16 that havebeen proposed to contact transcription factors are marked with anasterisk (*).

FIG. 3 includes an alignment showing modifications that may beintroduced into the TAD interaction motif of ERF2 to produce a variantERF2 interaction motif. The sequences of the native ERF2 and VP16interaction motifs are listed for comparison. The direct contacts arehighlighted.

FIG. 4 includes an alignment showing modifications that may beintroduced into the TAD interaction motif of PTI4 to produce a variantPTI4 interaction motif. The sequences of the native PTI4 and VP16interaction motifs are listed for comparison. The direct contacts arehighlighted.

FIG. 5 includes an alignment showing modifications that may beintroduced into the TAD interaction motif of AtERF1 to produce a variantAtEFR1 interaction motif. The sequences of the native AtERF1 and VP 16interaction motifs are listed for comparison. The direct contacts arehighlighted.

FIG. 6 includes an alignment showing modifications that may beintroduced into the TAD interaction motif of ORCA2 to produce a variantORCA2 interaction motif. The sequences of the native ORCA2 and VP16interaction motifs are listed for comparison. The direct contacts arehighlighted.

FIG. 7 includes an alignment showing modifications that may beintroduced into the TAD interaction motif of DREB1A to produce a variantDREB1A interaction motif. The sequences of the native DREB1A and VP 16interaction motifs are listed for comparison. The direct contacts arehighlighted.

FIG. 8 includes an alignment showing modifications that may beintroduced into the TAD interaction motif of CBF1 to produce a variantCBF1 interaction motif. The sequences of the native CBF1 and VP 16interaction motifs are listed for comparison. The direct contacts arehighlighted.

FIG. 9 includes an alignment showing modifications that may beintroduced into the TAD interaction motif of DOF1 to produce a variantDOF1 interaction motif. The sequences of the native DOF1 and VP16interaction motifs are listed for comparison. The direct contacts arehighlighted.

FIG. 10 includes a map of yeast integration vector, pHO-zBG-MEL1, whichcontains the HAS (High Affinity Site) ZFP binding sites upstream of aMEL1 reporter gene. The vector was targeted to the S. cerevisiae HOlocus, and contained a KanMX resistance gene for selection in both yeastand bacteria.

FIG. 11 includes a graphical illustration of the expression levels ofthe Mel1 reporter gene that resulted in yeast from activation by thedifferent plant transactivation interaction motifs.

FIG. 12 includes a map of plasmid pDAB9897: Arabidopsis thaliana actin-2promoter containing 8 tandem zinc finger (Z6) binding sites 548-749 basepairs upstream of transcriptional start site driving a gus reporter geneused for testing plant transactivation interaction motifs zinc fingerfusion proteins. The binary vector also contains an A. thalianaubiquitin-10 promoter driving a pat selectable marker for targetreporter plant event production.

FIG. 13 includes a map of plasmid pDAB107881.

FIG. 14 includes a map of plasmid pDAB107882.

FIG. 15 includes a map of plasmid pDAB107883.

FIG. 16 includes a map of plasmid pDAB107884.

FIG. 17 includes a map of plasmid pDAB107885.

FIG. 18 includes a map of plasmid pDAB107886.

FIG. 19 includes a map of plasmid pDAB107887.

FIG. 20 includes a map of plasmid pDAB106272.

FIG. 21 includes a map of plasmid pDAB106238.

FIG. 22 includes a map of plasmid pDAB106273.

FIG. 23 includes a map of plasmid pDAB106274.

FIG. 24 includes a map of plasmid pDAB106275.

FIG. 25 includes a map of plasmid pDAB106276.

FIG. 26 includes a map of plasmid pDAB106277.

FIG. 27 includes a map of plasmid pDAB106278.

FIG. 28 includes a map of plasmid pDAB106279.

FIG. 29 includes a graphical representation of the mean and standarddeviation (diamonds) and the quartiles (lines and boxes) of the gustranscript level normalized by endogenous gene expression level for thedifferent plant transactivation interaction motif treatments. Activationof the gus reporter gene from different plant transactivationinteraction motifs was compared to an empty vector control and theactivation of the domain II subunit of the VP 16 protein.

FIG. 30 includes a map of plasmid pGalGUS: Six tandem Gal4 binding sitesfused to an A. thaliana actin-2 promoter driving a gus reporter gene areused for testing plant transactivation interaction motifs fused to theGal4 binding protein. The binary vector also contains an A. thalianaubiquitin-10 promoter driving a pat selectable marker for targetreporter plant event production.

FIG. 31 includes a map of plasmid pTALGUS: Eight tandem UPA-Boxconsensus binding sites fused to an A. thaliana actin-2 promoter drivinga gus reporter gene are used for testing plant transactivationinteraction motifs fused to the TAL binding protein. The binary vectoralso contains an A. thaliana ubiquitin-10 promoter driving a patselectable marker for target reporter plant event production.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases, asdefined in 37 C.F.R. §1.822. Only one strand of each nucleic acidsequence is shown, but the complementary strand is understood to beincluded by any reference to the displayed strand. In the accompanyingsequence listing:

SEQ ID NO: 1 shows a VP16 plant transactivation domain containing aninteraction motif (underlined):

GMTHDPVSYGALDVDDFEFEQMFTDALGIDDFGG

SEQ ID NO: 2 shows a ERF2 plant transactivation domain containing aninteraction motif (underlined):

NDSEDMLVYGLLKDAFHFDTSSSDLSCLFDFPA

SEQ ID NO: 3 shows a PTI4 plant transactivation domain containing aninteraction motif (underlined):

CLTETWGDLPLKVDDSEDMVIYGLLKDALSVGWSPFSFTAG

SEQ ID NO: 4 shows a AtERF1 plant transactivation domain containing aninteraction motif (underlined):

CFTESWGDLPLKENDSEDMLVYGILNDAFHGG

SEQ ID NO: 5 shows a ORCA2 plant transactivation domain containing aninteraction motif (underlined):

FNENCEEIISPNYASEDLSDIILTDIFKDQDNYEDE

SEQ ID NO: 6 shows a DREB1A plant transactivation domain containing aninteraction motif (underlined):

GFDMEETLVEAIYTAEQSENAFYMHDEAMFEMPSLLANMAEGM

SEQ ID NO: 7 shows a CBF1 plant transactivation domain containing aninteraction motif (underlined):

EQSEGAFYMDEETMFGMPTLLDNMAEG

SEQ ID NO: 8 shows a DOF1 plant transactivation domain containing aninteraction motif (underlined):

SAGKAVLDDEDSFVWPAASFDMGACWAGAGFAD

SEQ ID NO: 9 shows subdomain II of a VP16 transactivation domain, whichis the interaction motif within SEQ ID NO: 1:

DDFEFEQMFTD

SEQ ID NO: 10 shows a ERF2 plant transactivation domain interactionmotif:

DAFHFDTSSSD

SEQ ID NO: 11 shows a PTI4 plant transactivation domain interactionmotif:

DDSEDMVIYGLLKD

SEQ ID NO: 12 shows a AtERF1 plant transactivation domain interactionmotif:

ENDSEDMLV

SEQ ID NO: 13 shows a ORCA2 plant transactivation domain interactionmotif:

EDLSDIILTD

SEQ ID NO: 14 shows a DREB1A plant transactivation domain interactionmotif:

ENAFYMHDEAMFEMP

SEQ ID NO: 15 shows a CBF1 plant transactivation domain interactionmotif:

DEETMFGMP

SEQ ID NO: 16 shows a DOF1 plant transactivation domain interactionmotif:

EDSFVWPAASFD

SEQ ID NOs: 17-22 show variant ERF2 plant transactivation domaininteraction motif sequences.

SEQ ID NOs: 23-28 show variant PTI4 plant transactivation domaininteraction motif sequences.

SEQ ID NOs: 29-34 show variant AtERF1 plant transactivation domaininteraction motif sequences.

SEQ ID NOs: 35-40 show variant ORCA2 plant transactivation domaininteraction motif sequences.

SEQ ID NOs: 41-46 show variant DREB1A plant transactivation domaininteraction motif sequences.

SEQ ID NOs: 47-52 show variant CBF1 plant transactivation domaininteraction motif sequences.

SEQ ID NOs: 53-58 show variant DOF1 plant transactivation domaininteraction motif sequences.

SEQ ID NOs: 59-66 show primers used for the construction of plasmid,pHO-zBG-MEL1.

SEQ ID NO: 67 shows a Z6 DNA binding domain polynucleotide sequence:

TGTGGTGGGAGAGGAGGGTGG

SEQ ID NO: 68 shows an 8× tandem repeat sequence of a Z6 DNA bindingdomain:

GGTGTGGTGGGAGAGGAGGGTGGGAGTGTGGTGGGAGAGGAGGGTGGCTCTGTGGTGGGAGAGGAGGGTGGAGATGTGGTGGGAGAGGAGGGTGGTCTTGTGGTGGGAGAGGAGGGTGGGGATGTGGTGGGAGAGGAGGGTGGCCTTGTGGTGGGAGAGGAGGGTGGAGGTGTGGTGGGAGAGGAGGGTGGCTTAA GCCGC

SEQ ID NOs: 69-74 show primers and probes used in pat and pal HP assays.

SEQ ID NOs: 75-78 show primers used for PCR analysis of PTUs in tobacco.

SEQ ID NO: 79 shows a synthetic nucleotide sequence encoding a nativeplant transactivation domain interaction motif from VP 16 that was fusedto a Z6 Zinc Finger binding Protein:

GGCATGACCCATGATCCTGTGTCTTATGGAGCCTTGGATGTTGATGACTTTGAGTTTGAGCAGATGTTCACAGATGCACTGGGCATCGATGACTTTGG TGGA

SEQ ID NO: 80 shows a synthetic nucleotide sequence (v3) encoding anative plant transactivation domain interaction motif from ERF2 that wasfused to a Z6 Zinc Finger binding Protein:

AATGACTCTGAGGACATGCTGGTGTATGGTTTGCTCAAGGATGCCTTTCACTTTGACACCTCCAGCTCAGACCTCTCCTGCCTCTTTGACTTCCCAGC C

SEQ ID NO: 81 shows a synthetic nucleotide sequence (v3) encoding anative plant transactivation interaction motif from PTI4 that was fusedto a Z6 Zinc Finger binding Protein:

TGCCTGACAGAAACTTGGGGAGACTTGCCTCTCAAGGTTGATGACTCTGAGGACATGGTGATCTATGGTCTGTTGAAGGATGCACTCTCAGTGGGGTGGTCCCCATTCTCTTTCACGGCTGGT

SEQ ID NO: 82 shows a synthetic nucleotide sequence (v3) encoding anative plant transactivation domain interaction motif from AtERF1 thatwas fused to a Z6 Zinc Finger binding Protein:

TGCTTCACGGAATCCTGGGGAGACCTTCCTTTGAAGGAGAATGACTCTGAGGACATGTTGGTGTACGGAATCCTCAATGATGCTTTTCATGGTGGC

SEQ ID NO: 83 shows a synthetic nucleotide sequence (v3) encoding anative plant transactivation domain interaction motif from ORCA2 thatwas fused to a Z6 Zinc Finger binding Protein:

TTCAATGAGAATTGTGAAGAAATCATCTCTCCAAACTACGCATCAGAGGACTTGTCTGACATCATCTTGACGGACATCTTCAAGGACCAAGACAACTA TGAGGATGAG

SEQ ID NO: 84 shows a synthetic nucleotide sequence (v3) encoding anative plant transactivation domain interaction motif from DREB1A thatwas fused to a Z6 Zinc Finger binding Protein:

GGCTTTGACATGGAAGAAACATTGGTGGAGGCCATCTACACTGCTGAACAGAGCGAGAATGCCTTCTACATGCATGATGAGGCAATGTTTGAGATGCCATCTCTTCTGGCCAACATGGCTGAGGGAATG

SEQ ID NO: 85 shows a synthetic nucleotide sequence (v3) encoding anative plant transactivation domain interaction motif from CBF1 that wasfused to a Z6 Zinc Finger binding Protein:

GAACAGTCAGAAGGTGCTTTCTACATGGATGAAGAGACCATGTTTGGGATGCCAACCCTTCTGGATAACATGGCAGAGGGA

SEQ ID NO: 86 shows a synthetic nucleotide sequence (v3) encoding anative plant transactivation domain interaction motif from DOF1 that wasfused to a Z6 Zinc Finger binding Protein:

TCAGCTGGGAAGGCAGTCTTGGATGATGAGGACAGCTTTGTTTGGCCTGCTGCATCCTTTGACATGGGTGCCTGCTGGGCTGGAGCTGGCTTTGCTGA C

SEQ ID NO: 87 shows a synthetic nucleotide sequence (v2) encoding anexemplary variant plant transactivation domain interaction motif fromERF2 that was fused to a Z6 Zinc Finger binding Protein:

AATGACTCTGAGGACATGCTGGTGTATGGTTTGCTCAAGGATGATTTCCACTTTGAGACAATGTTCTCAGACCTGTCCTGCCTCTTTGACTTCCCAGC C

SEQ ID NO: 88 shows a synthetic nucleotide sequence (v2) encoding anexemplary variant plant transactivation domain interaction motif fromPTI4 that was fused to a Z6 Zinc Finger binding Protein:

TGCCTGACAGAAACTTGGGGAGACTTGCCTCTCAAGGTTGATGACTTTGAGTTTGAGATGATGTTCACAGATGCACTCTCAGTGGGGTGGTCCCCATT CTCTTTCACGGCTGGT

SEQ ID NO: 89 shows a synthetic nucleotide sequence (v2) encoding anexemplary variant plant transactivation domain interaction motif fromAtERF1 that was fused to a Z6 Zinc Finger binding Protein:

TGCTTCACGGAATCCTGGGGAGACCTTCCTTTGAAGGAGAATGACTTTGAGTTTGAAATGTTCACAGATTACGGAATCCTCAATGATGCTTTTCATGG TGGC

SEQ ID NO: 90 shows a synthetic nucleotide sequence (v2) encoding anexemplary variant plant transactivation domain interaction motif fromORCA2 that was fused to a Z6 Zinc Finger binding Protein:

TTCAATGAGAATTGTGAAGAAATCATCTCTCCAAACTACGCATCAGAGGACTTTGATCTTGAGATGTTGACGGACATCTTCAAGGACCAAGACAACTA TGAGGATGAG

SEQ ID NO: 91 shows a synthetic nucleotide sequence (v2) encoding anexemplary variant plant transactivation domain interaction motif fromDREB1A that was fused to a Z6 Zinc Finger binding Protein:

GGCTTTGACATGGAAGAAACATTGGTGGAGGCCATCTACACTGCTGAACAGAGCGAGGACTTTGAGTTTGAAGCAATGTTCATGGATTCTCTTCTGGC CAACATGGCTGAGGGAATG

SEQ ID NO: 92 shows a synthetic nucleotide sequence (v2) encoding anexemplary variant plant transactivation domain interaction motif fromCBF1 that was fused to a Z6 Zinc Finger binding Protein:

GAACAGTCAGAAGGTGCTTTCTACATGGATGACTTTGAGTTCGAGACAATGTTCATGGACACCCTTCTGGATAACATGGCAGAGGGA

SEQ ID NO: 93 shows a synthetic nucleotide sequence (v2) encoding anexemplary variant plant transactivation domain interaction motif fromDOF1 that was fused to a Z6 Zinc Finger binding Protein:

TCAGCTGGGAAGGCAGTCTTGGATGATGAGGACTTTGAGTTTGAAGCCATGTTCACGGACATGGGTGCCTGCTGGGCTGGAGCTGGCTTTGCTGAC

SEQ ID NOs: 94-98 show primers and probes used in gus and BYEEF HPassays.

SEQ ID NO: 99 shows a tandem repeat sequence taken from the consensusbinding sequence of AVRBS3-inducible genes, and termed the UPA DNAbinding domain:

TATATAAACCTNNCCCTCT

SEQ ID NO: 100 shows an exemplary synthetic transactivation domaincomprising a ERF2 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVDAFHFDTSSSDALGIDDFGG

SEQ ID NO: 101 shows an exemplary synthetic transactivation domaincomprising a PTI4 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVDDSEDMVIYGLLKDALGIDDFGG

SEQ ID NO: 102 shows an exemplary synthetic transactivation domaincomprising a AtERF1 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVENDSEDMLVALGIDDFGG

SEQ ID NO: 103 shows an exemplary synthetic transactivation domaincomprising a ORCA2 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVEDLSDIILTDALGIDDFGG

SEQ ID NO: 104 shows an exemplary synthetic transactivation domaincomprising a DREB1A plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVENAFYMHDEAMFEMPALGIDDFGG

SEQ ID NO: 105 shows an exemplary synthetic transactivation domaincomprising a CBF1 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVDEETMFGMPALGIDDFGG

SEQ ID NO: 106 shows an exemplary synthetic transactivation domaincomprising a DOF1 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVEDSFVWPAASFDALGIDDFGG

SEQ ID NO: 107 shows an exemplary synthetic transactivation domaincomprising a variant ERF2 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVDDFHFETMFSDALGIDDFGG

SEQ ID NO: 108 shows a further exemplary synthetic transactivationdomain comprising a variant ERF2 plant transactivation domaininteraction motif (underlined):

NDSEDMLVYGLLKDDFHFETMFSDLSCLFDFPA

SEQ ID NO: 109 shows an exemplary synthetic transactivation domaincomprising a variant PTI4 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVDDFEFEMMFTDALGIDDFGG

SEQ ID NO: 110 shows a further exemplary synthetic transactivationdomain comprising a variant PTI4 plant transactivation domaininteraction motif (underlined):

CLTETWGDLPLKVDDFEFEMMFTDALSVGWSPFSFTAG

SEQ ID NO: 111 shows an exemplary synthetic transactivation domaincomprising a variant AtERF1 plant transactivation domain interactionmotif (underlined):

GMTHDPVSYGALDVENDFEFEMFTDALGIDDFGG

SEQ ID NO: 112 shows a further exemplary synthetic transactivationdomain comprising a variant AtERF1 plant transactivation domaininteraction motif (underlined):

CFTESWGDLPLKENDFEFEMFTDYGILNDAFHGG

SEQ ID NO: 113 shows an exemplary synthetic transactivation domaincomprising a variant ORCA2 plant transactivation domain interactionmotif (underlined):

GMTHDPVSYGALDVEDFDLEMLTDALGIDDFGG

SEQ ID NO: 114 shows a further exemplary synthetic transactivationdomain comprising a variant ORCA2 plant transactivation domaininteraction motif (underlined):

FNENCEEIISPNYASEDFDLEMLTDIFKDQDNYEDE

SEQ ID NO: 115 shows an exemplary synthetic transactivation domaincomprising a variant DREB1A plant transactivation domain interactionmotif (underlined):

GMTHDPVSYGALDVEDFEFEAMFMDALGIDDFGG

SEQ ID NO: 116 shows a further exemplary synthetic transactivationdomain comprising a variant DREB1A plant transactivation domaininteraction motif (underlined):

GFDMEETLVEAIYTAEQSEDFEFEAMFMDSLLANMAEGM

SEQ ID NO: 117 shows an exemplary synthetic transactivation domaincomprising a variant CBF1 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVDDFEFETMFMDALGIDDFGG

SEQ ID NO: 118 shows a further exemplary synthetic transactivationdomain comprising a variant CBF1 plant transactivation domaininteraction motif (underlined):

EQSEGAFYMDDFEFETMFMDTLLDNMAEG

SEQ ID NO: 119 shows an exemplary synthetic transactivation domaincomprising a variant DOF1 plant transactivation domain interaction motif(underlined):

GMTHDPVSYGALDVEDFEFEAMFTDALGIDDFGG

SEQ ID NO: 120 shows a further exemplary synthetic transactivationdomain comprising a variant DOF1 plant transactivation domaininteraction motif (underlined):

SAGKAVLDDEDFEFEAMFTDMGACWAGAGFAD

SEQ ID NOs: 121-127 show variant plant transactivation domaininteraction motif sequences.

DETAILED DESCRIPTION I. Overview of Several Embodiments

Disclosed herein are novel plant transactivation domains (TADs), TADinteraction motifs, and synthetic variants of the foregoing that may beuseful as transcriptional activators, and that may be fused in asynthetic transcriptional activator fusion protein with a DNA-bindingpolypeptide for transcriptional activation of a gene of interest.Particular novel plant TADs and TAD interaction motifs disclosed hereinhave been isolated from the plant proteins, ERF2; PTI4; AtERF1; ORCA2;DREB1A; CBF1; and DOF1. Synthetic transcriptional activator fusionproteins comprising novel plant TAD and/or TAD interaction motif asdescribed herein may be utilized in particular embodiments to increase(e.g., initiate) gene expression in a variety of cells (e.g., yeastcells and plant cells), and for virtually any gene.

Transactivation domains are functionally autonomous; i.e., a single TADcan regulate transcription when fused to one of many differentheterologous DNA-binding domains, and when tethered at differentpositions in a promoter region. Hall and Struhl (2002), supra. TADs arebelieved to share little primary sequence homology and adopt a definedstructure only upon binding to a target. Sigler (1988), supra. Thoughacidic and hydrophobic residues within the TADs are thought to beimportant (see, e.g., Cress and Triezenberg (1991), supra), thecontribution of individual residues to activity is believed to be small.Hall and Struhl (2002), supra.

It is difficult to predict a priori if a synthetic transactivationdomain interaction motif will function to initiate or enhance expressionin a plant cell. This unpredictability may be at least in part aconsequence of the fact that some TADs are very strong transactivatorsthat may result in “squelching” (e.g., by titrating components of thecellular transcriptional machinery) as a function both of itsintracellular concentration and the strength of its TADs. See, e.g.,U.S. Pat. No. 6,271,341 (mutant VP16 TADs with graded gene regulation).

Disclosed herein is the unexpected finding that certain novel plant TADsand TAD interaction motifs sharing sequence homology with the VP16 TADconfer very different levels of regulation upon genes under theircontrol. Using a generalizable strategy for “swapping” TADs to producesynthetic transcriptional activator fusion proteins, it was surprisinglyfound that novel TADs and TAD interaction motifs isolated from PTI4,DREB1A, ERF2, and CBF1 are able to provide greater increases in genetranscription in a plant cell than is provided by VP16, which isrecognized in the art as being a very good transactivator. It was alsofound that novel TADs and TAD interaction motifs from AtERF1, ORCA2, andDOF1 provide lesser increases in gene transcription.

Also disclosed herein is the unexpected finding that variant TADs andTAD interaction motifs comprising very few and minor amino acid changeswith regard to the native sequence may provide further enhancement ortuning of the gene regulatory properties exhibited by the native TAD.For example, it was surprisingly found that variant ERF2 and CBF1 TADinteraction motifs lead to significantly greater transcription of a geneunder its control than the corresponding native interaction motif inplants.

II. Abbreviations

chs chalcone synthase gene

HAS high affinity site

HP hydrolysis probe

HSV Herpes Simplex Virus

MS Murashige and Skoog

PNPG p-nitrophenyl-alpha-D-glucopyranoside

PTU plant transcriptional unit

SSC saline-sodium citrate

TAD transactivation domain

TBP TATA-binding protein

T-DNA transfer DNA

TFIIB transcription factor IIB

TFIIH transcription factor IIH

T_(i) tumor-inducing (plasmids derived from A. tumefaciens)

UAS upstream activation sequence

VP16 Herpes Simplex Virion Protein 16

III. Terms

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Endogenous: As used herein, the term “endogenous” refers to substances(e.g., nucleic acid molecules and polypeptides) that originate fromwithin a particular organism, tissue, or cell. For example, an“endogenous” polypeptide expressed in a plant cell may refer to apolypeptide that is normally expressed in cells of the same type fromnon-genetically engineered plants of the same species. Likewise, an“endogenous” nucleic acid comprised in a plant cell may refer to anucleic acid (e.g., genomic DNA) that is normally found in cells of thesame type from non-genetically engineered plants of the same species.

Expression: As used herein, “expression” of a coding sequence (forexample, a gene or a transgene) refers to the process by which the codedinformation of a nucleic acid transcriptional unit (including, e.g.,genomic DNA or cDNA) is converted into an operational, non-operational,or structural part of a cell (e.g., a protein). Gene expression can beinfluenced by external signals; for example, exposure of a cell, tissue,or organism to an agent that increases or decreases expression of a genecomprised therein. Expression of a gene can also be regulated anywherein the pathway from DNA to RNA to protein. Regulation of gene expressionoccurs, for example, through controls acting on transcription,translation, RNA transport and processing, degradation of intermediarymolecules such as mRNA, or through activation, inactivation,compartmentalization, or degradation of specific protein molecules afterthey have been made, or by combinations of any of the foregoing. Geneexpression can be measured at the RNA level or the protein level bymethods known in the art, including, without limitation, Northern blot,RT-PCR, Western blot, and in vitro, in situ, or in vivo protein activityassay(s).

Increase expression: As used herein, the term “increase expression”refers to initiation of expression, as well as to a quantitativeincrease in the amount of an expression product produced from a templateconstruct. In some embodiments, a polypeptide comprising a TAD may beused to “increase expression” from a nucleic acid. In such embodiments,the increase in expression may be determined by comparison with theamount of expression product produced in a control (e.g., from theconstruct in the absence of the protein comprising the planttransactivation domain).

Fusion protein: As used herein, the term “fusion protein” refers to amolecule comprising at least two operatively linked polypeptides. Incertain examples, the two operatively linked polypeptides may benormally expressed as part of different gene products (e.g., indifferent organisms). In further examples, the at least two operativelylinked polypeptides may be derived from polypeptides normally expressedas part of different gene products. The operatively linked polypeptidespresent in a fusion protein described herein typically interact with atleast one target protein or nucleic acid in a cell wherein the fusionprotein is to be expressed. For example, an operatively linkedpolypeptide may interact with one or more transcription factor(s) orproteinaceous element(s) of the cellular transcription machinery, or itmay interact with a specific polynucleotide or structural element of anucleic acid.

Heterologous: As used herein, the term “heterologous” refers tosubstances (e.g., nucleic acid molecules and polypeptides) that do notoriginate from within a particular organism, tissue, or cell. Forexample, a “heterologous” polypeptide expressed in a plant cell mayrefer to a polypeptide that is not normally expressed in cells of thesame type from non-genetically engineered plants of the same species(e.g., a polypeptide that is expressed in different cells of the sameorganism or cells of a different organism).

Isolated: An “isolated” biological component (such as a nucleic acid orprotein) has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs (e.g., otherchromosomal and extra-chromosomal DNA and RNA, and proteins), whileeffecting a chemical or functional change in the component (e.g., anucleic acid may be isolated from a chromosome by breaking chemicalbonds connecting the nucleic acid to the remaining DNA in thechromosome). Nucleic acid molecules and proteins that have been“isolated” may include nucleic acid molecules and proteins purified bystandard purification methods. The term embraces nucleic acids andproteins prepared by recombinant expression in a host cell, as well aschemically-synthesized nucleic acid molecules, proteins, and peptides.

Nucleic acid molecule: As used herein, the term “nucleic acid molecule”may refer to a polymeric form of nucleotides, which may include bothsense and anti-sense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. A nucleotide may refer to aribonucleotide, deoxyribonucleotide, or a modified form of either typeof nucleotide. A “nucleic acid molecule” as used herein is synonymouswith “nucleic acid” and “polynucleotide.” A nucleic acid molecule isusually at least 10 bases in length, unless otherwise specified. Theterm includes single- and double-stranded forms of DNA. A nucleic acidmolecule can include either or both naturally occurring and modifiednucleotides linked together by naturally occurring and/or non-naturallyoccurring nucleotide linkages.

An “exogenous” molecule is a molecule that is not native to a specifiedsystem (e.g., a germplasm, variety, elite variety, and/or plant) withrespect to nucleotide sequence and/or genomic location for apolynucleotide, and with respect to amino acid sequence and/or cellularlocalization for a polypeptide. In embodiments, exogenous orheterologous polynucleotides or polypeptides may be molecules that havebeen artificially supplied to a biological system (e.g., a plant cell, aplant gene, a particular plant species or variety, and/or a plantchromosome) and are not native to that particular biological system.Thus, the designation of a nucleic acid as “exogenous” may indicate thatthe nucleic acid originated from a source other than anaturally-occurring source, or it may indicate that the nucleic acid hasa non-natural configuration, genetic location, or arrangement ofelements.

In contrast, for example, a “native” or “endogenous” nucleic acid is anucleic acid (e.g., a gene) that does not contain a nucleic acid elementother than those normally present in the chromosome or other geneticmaterial on which the nucleic acid is normally found in nature. Anendogenous gene transcript is encoded by a nucleotide sequence at itsnatural chromosomal locus, and is not artificially supplied to the cell.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications (e.g., uncharged linkages: for example, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.;charged linkages: for example, phosphorothioates, phosphorodithioates,etc.; pendent moieties: for example, peptides; intercalators: forexample, acridine, psoralen, etc.; chelators; alkylators; and modifiedlinkages: for example, alpha anomeric nucleic acids, etc.). The term“nucleic acid molecule” also includes any topological conformation,including single-stranded, double-stranded, partially duplexed,triplexed, hairpinned, circular, and padlocked conformations.

Some embodiments employ a particular form of nucleic acid, anoligonucleotide. Oligonucleotides are relatively short nucleic acidmolecules, typically comprising 50 or fewer nucleobases (though someoligonucleotides may comprise more than 50). An oligonucleotide may beformed by cleavage (e.g., restriction digestion) of a longer nucleicacid comprising the oligonucleotide sequence, or it may be chemicallysynthesized, in a sequence-specific manner, from individual nucleosidephosphoramidites.

An oligonucleotide may be used as a probe sequence to detect a nucleicacid molecule comprising a particular nucleotide sequence. According tothe foregoing, an oligonucleotide probe may be prepared synthetically orby cloning. Suitable cloning vectors are known to those of skill in theart. An oligonucleotide probe may be labeled or unlabeled. A widevariety of techniques exist for labeling nucleic acid molecules,including, for example and without limitation, radiolabeling by nicktranslation; random priming; and tailing with terminal deoxytransferase,where the nucleotides employed are labeled, for example, withradioactive ³²P. Other labels that may be used include, for example andwithout limitation: fluorophores; enzymes; enzyme substrates; enzymecofactors; and enzyme inhibitors. Alternatively, the use of a label thatprovides a detectable signal, by itself or in conjunction with otherreactive agents, may be replaced by ligands to which receptors bind,where the receptors are labeled (for example, by the above-indicatedlabels) to provide detectable signals, either by themselves, or inconjunction with other reagents. See, e.g., Leary et al. (1983) Proc.Natl. Acad. Sci. USA 80:4045-9.

Some embodiments of the invention include a polynucleotide that is“specifically hybridizable” or “specifically complementary” to anucleotide target sequence. “Specifically hybridizable” and“specifically complementary” are terms that indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe polynucleotide and the nucleic acid molecule comprising theparticular nucleotide target sequence. A nucleic acid molecule need notbe 100% complementary to its target sequence to be specificallyhybridizable. A nucleic acid molecule is specifically hybridizable whenthere is a sufficient degree of complementarity to avoid non-specificbinding of the nucleic acid to non-target sequences under conditionswhere specific binding is desired, for example, under stringenthybridization conditions.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg⁺⁺ concentration) of thehybridization buffer will contribute to the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are known to those of ordinary skill in the art, and arediscussed, for example, in Sambrook et al. (ed.) Molecular Cloning: ALaboratory Manual, 2″ ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames andHiggins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.Further detailed instruction and guidance with regard to thehybridization of nucleic acids may be found, for example, in Tijssen,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” in Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2, Elsevier, NY, 1993; and Ausubel et al., Eds., CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, NY, 1995.

As used herein, “stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 25% mismatch betweenthe hybridization molecule and the DNA target. “Stringent conditions”include further particular levels of stringency. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize; and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize.

In particular embodiments, stringent conditions are hybridization for 1hour at 65° C. in a PerfectHyb™ plus hybridization buffer(Sigma-Aldrich), followed by 40 minute sequential washes at 65° C. in0.1×SSC/0.1% SDS.

Operably linked nucleotide sequences: A first nucleotide sequence is“operably linked” with or to a second nucleotide sequence when the firstnucleotide sequence is in a functional relationship with the secondnucleotide sequence. For instance, a promoter is operably linked to acoding sequence if the promoter affects the transcription or expressionof the coding sequence. When recombinantly produced, operably linkednucleotide sequences are generally contiguous and, where necessary tojoin two protein-coding regions, in the same reading frame. However,nucleotide sequences need not be contiguous to be operably linked.

The term, “operably linked,” when used in reference to a gene regulatorysequence and a coding sequence, means that the regulatory sequenceaffects the expression of the linked coding sequence. “Regulatorysequences,” or “control elements,” refer to nucleotide sequences thatinfluence the timing and level/amount of transcription, RNA processingor stability, or translation of the associated coding sequence.Conventional regulatory sequences may include 5′ untranslated regions;promoters; translation leader sequences; introns; enhancers; stem-loopstructures; repressor binding sequences; termination sequences;polyadenylation recognition sequences; etc. Particular regulatorysequences may be located upstream and/or downstream of a coding sequenceoperably linked thereto. Also, particular regulatory sequences operablylinked to a coding sequence may be located on the associatedcomplementary strand of a double-stranded nucleic acid molecule.Elements that may be “operably linked” to a coding sequence are notlimited to promoters or other conventional regulatory sequences. Forexample, in some embodiments, the DNA-binding domain of a transactivatorprotein may bind to a nucleotide sequence that is proximal to a promoteror other regulatory region, such that the transactivator protein mayinteract with the promoter or other regulatory region, or a moleculebound thereto (e.g., a transcription factor) to affect transcription. Insuch examples, the nucleotide sequence to which the transactivatorprotein binds through its DNA-binding domain is “operably linked” to thecoding sequence under the control of the promoter or other regulatorysequence.

Operatively linked polypeptides: As used herein with regard topolypeptides, the term “operatively linked” refers to at least twopolypeptides that are connected in a single molecule (e.g., a fusionprotein), and in such a manner that each polypeptide can serve itsintended function. Typically, the at least two polypeptides arecovalently attached through peptide bonds. A fusion protein comprisingoperatively linked polypeptides may be produced by standard recombinantDNA techniques. For example, a DNA molecule encoding a first polypeptidemay be ligated to another DNA molecule encoding a second polypeptide,and the resultant hybrid DNA molecule may be expressed in a host cell toproduce a fusion protein comprising the first and second polypeptides.In particular examples, the two DNA molecules may be ligated to eachother in a 5′ to 3′ orientation, such that, after ligation, thetranslational frame of the encoded polypeptides is not altered (i.e.,the DNA molecules are ligated to each other in-frame).

Promoter: As used herein, the term “promoter” refers to a region of DNAthat may be upstream from the start of transcription, and that may beinvolved in recognition and binding of RNA polymerase and other proteinsto effect transcription. A promoter may be operably linked to a codingsequence for expression in a cell, or a promoter may be operably linkedto a nucleotide sequence encoding a signal sequence which may beoperably linked to a coding sequence for expression in a cell. A “plantpromoter” may be a promoter capable of initiating transcription in aplant cell.

Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, for exampleand without limitation, leaves, roots, seeds, fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred.” Promoters which initiate transcription only incertain tissues are referred to as “tissue-specific.” A “celltype-specific” promoter primarily effects transcription in certain celltypes in one or more organs, for example and without limitation, invascular cells in roots or leaves. Exemplary tissue-specific ortissue-preferred promoters include, but are not limited to: Aroot-preferred promoter, such as that from the phaseolin gene; aleaf-specific and light-induced promoter such as that from cab orrubisco; an anther-specific promoter such as that from LAT52; apollen-specific promoter such as that from Zm13; and amicrospore-preferred promoter such as that from apg.

An “inducible” promoter may be a promoter which may be underenvironmental control. See Ward et al. (1993) Plant Mol. Biol.22:361-366. Examples of environmental conditions that may initiatetranscription by inducible promoters include, for example and withoutlimitation, anaerobic conditions and the presence of light. With aninducible promoter, the rate of transcription increases in response toan inducing agent. Exemplary inducible promoters include, but are notlimited to: Promoters from the ACEI system that responds to copper; In2gene from maize that responds to benzenesulfonamide herbicide safeners;Tet repressor from Tn10; and the inducible promoter from a steroidhormone gene, the transcriptional activity of which may be induced by aglucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci.USA 88:0421).

Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that may be active under mostenvironmental conditions. Exemplary constitutive promoters include, butare not limited to: Promoters from plant viruses, such as the ³⁵Spromoter from CaMV; promoters from rice actin genes; ubiquitinpromoters; pEMU; MAS; maize H3 histone promoter; and the ALS promoter,Xbal/NcoI fragment 5′ to the Brassica napus ALS3 structural gene (or anucleotide sequence similarity to said Xbal/NcoI fragment)(International PCT Publication No. WO 96/30530).

Any of the foregoing constitutive and non-constitutive promoters may beutilized in some embodiments of the invention. For example, a gene to beregulated by the activity of a synthetic transcriptional activatorfusion protein may be provided (e.g., in a host cell), wherein the geneis operably linked to a promoter.

Sequence identity: The term “sequence identity” or “identity,” as usedherein in the context of two nucleic acid or polypeptide sequences, mayrefer to the residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.

As used herein, the term “percentage of sequence identity” may refer tothe value determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences, and amino acid sequences) over a comparisonwindow, wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleotide oramino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in, for example:Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch(1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higginsand Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearsonet al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMSMicrobiol. Lett. 174:247-50. A detailed consideration of sequencealignment methods and homology calculations can be found in, forexample, Altschul et al. (1990) J. Mol. Biol. 215:403-10.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990)) is available fromseveral sources, including the National Center for BiotechnologyInformation (Bethesda, Md.), and on the internet, for use in connectionwith several sequence analysis programs. A description of how todetermine sequence identity using this program is available on theinternet under the “help” section for BLAST™. For comparisons of nucleicacid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn)program may be employed using the default parameters. Nucleic acidsequences with even greater similarity to the reference sequences willshow increasing percentage identity when assessed by this method.

As used herein with regard to nucleotide sequences, the term“substantially identical” may refer to sequences that are more than 85%identical. For example, a substantially identical nucleotide sequencemay be at least 85.5%; at least 86%; at least 87%; at least 88%; atleast 89%; at least 90%; at least 91%; at least 92%; at least 93%; atleast 94%; at least 95%; at least 96%; at least 97%; at least 98%; atleast 99%; or at least 99.5% identical to the reference sequence.

Conservative substitution: As used herein, the term “conservativesubstitution” refers to a substitution where an amino acid residue issubstituted for another amino acid in the same class. A non-conservativeamino acid substitution is one where the residues do not fall into thesame class, for example, substitution of a basic amino acid for aneutral or non-polar amino acid. Classes of amino acids that may bedefined for the purpose of performing a conservative substitution areknown in the art.

In some embodiments, a conservative substitution includes thesubstitution of a first aliphatic amino acid for a second, differentaliphatic amino acid. For example, if a first amino acid is one of Gly;Ala; Pro; Ile; Leu; Val; and Met, the first amino acid may be replacedby a second, different amino acid selected from Gly; Ala; Pro; Ile; Leu;Val; and Met. In particular examples, if a first amino acid is one ofGly; Ala; Pro; Ile; Leu; and Val, the first amino acid may be replacedby a second, different amino acid selected from Gly; Ala; Pro; Ile; Leu;and Val. In particular examples involving the substitution ofhydrophobic aliphatic amino acids, if a first amino acid is one of Ala;Pro; Ile; Leu; and Val, the first amino acid may be replaced by asecond, different amino acid selected from Ala; Pro; Ile; Leu; and Val.

In some embodiments, a conservative substitution includes thesubstitution of a first aromatic amino acid for a second, differentaromatic amino acid. For example, if a first amino acid is one of His;Phe; Trp; and Tyr, the first amino acid may be replaced by a second,different amino acid selected from His; Phe; Trp; and Tyr. In particularexamples involving the substitution of uncharged aromatic amino acids,if a first amino acid is one of Phe; Trp; and Tyr, the first amino acidmay be replaced by a second, different amino acid selected from Phe;Trp; and Tyr.

In some embodiments, a conservative substitution includes thesubstitution of a first hydrophobic amino acid for a second, differenthydrophobic amino acid. For example, if a first amino acid is one ofAla; Val; Ile; Leu; Met; Phe; Tyr; and Trp, the first amino acid may bereplaced by a second, different amino acid selected from Ala; Val; Ile;Leu; Met; Phe; Tyr; and Trp. In particular examples involving thesubstitution of non-aromatic, hydrophobic amino acids, if a first aminoacid is one of Ala; Val; Ile; Leu; and Met, the first amino acid may bereplaced by a second, different amino acid selected from Ala; Val; Ile;Leu; and Met.

In some embodiments, a conservative substitution includes thesubstitution of a first polar amino acid for a second, different polaramino acid. For example, if a first amino acid is one of Ser; Thr; Asn;Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu, the first amino acidmay be replaced by a second, different amino acid selected from Ser;Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu. In particularexamples involving the substitution of uncharged, polar amino acids, ifa first amino acid is one of Ser; Thr; Asn; Gln; Cys; Gly; and Pro, thefirst amino acid may be replaced by a second, different amino acidselected from Ser; Thr; Asn; Gln; Cys; Gly; and Pro. In particularexamples involving the substitution of charged, polar amino acids, if afirst amino acid is one of His; Arg; Lys; Asp; and Glu, the first aminoacid may be replaced by a second, different amino acid selected fromHis; Arg; Lys; Asp; and Glu. In further examples involving thesubstitution of charged, polar amino acids, if a first amino acid is oneof Arg; Lys; Asp; and Glu, the first amino acid may be replaced by asecond, different amino acid selected from Arg; Lys; Asp; and Glu. Inparticular examples involving the substitution of positively charged(basic), polar amino acids, if a first amino acid is one of His; Arg;and Lys, the first amino acid may be replaced by a second, differentamino acid selected from His; Arg; and Lys. In further examplesinvolving the substitution of positively charged, polar amino acids, ifa first amino acid is Arg or Lys, the first amino acid may be replacedby the other amino acid of Arg and Lys. In particular examples involvingthe substitution of negatively charged (acidic), polar amino acids, if afirst amino acid is Asp or Glu, the first amino acid may be replaced bythe other amino acid of Asp and Glu. In particular examples involvingthe substitution of polar amino acids other than positively chargedpolar amino acids, if a first amino acid is one of Ser; Thr; Asn; Gln;Cys; Gly; Pro; Asp; and Glu, the first amino acid may be replaced by asecond, different amino acid selected from Ser; Thr; Asn; Gln; Cys; Gly;Pro; Asp; and Glu. In particular examples involving the substitution ofpolar amino acids other than negatively charged polar amino acids, if afirst amino acid is one of Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His;and Lys, the first amino acid may be replaced by a second, differentamino acid selected from Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His;and Lys.

In some embodiments, a conservative substitution includes thesubstitution of a first electrically neutral amino acid for a second,different electrically neutral amino acid. For example, if a first aminoacid is one of Gly; Ser; Thr; Cys; Asn; Gln; and Tyr, the first aminoacid may be replaced by a second, different amino acid selected fromGly; Ser; Thr; Cys; Asn; Gln; and Tyr.

In some embodiments, a conservative substitution includes thesubstitution of a first non-polar amino acid for a second, differentnon-polar amino acid. For example, if a first amino acid is one of Ala;Val; Leu; Ile; Phe; Trp; Pro; and Met, the first amino acid may bereplaced by a second, different amino acid selected from Ala; Val; Leu;Ile; Phe; Trp; Pro; and Met.

In many examples, the selection of a particular second amino acid to beused in a conservative substitution to replace a first amino acid may bemade in order to maximize the number of the foregoing classes to whichthe first and second amino acids both belong. Thus, if the first aminoacid is Ser (a polar, non-aromatic, and electrically neutral aminoacid), the second amino acid may be another polar amino acid (i.e., Thr;Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; or Glu); anothernon-aromatic amino acid (i.e., Thr; Asn; Gln; Cys; Gly; Pro; Arg; His;Lys; Asp; Glu; Ala; Ile; Leu; Val; or Met); or anotherelectrically-neutral amino acid (i.e., Gly; Thr; Cys; Asn; Gln; or Tyr).However, it may be preferred that the second amino acid in this case beone of Thr; Asn; Gln; Cys; and Gly, because these amino acids share allthe classifications according to polarity, non-aromaticity, andelectrical neutrality. Additional criteria that may optionally be usedto select a particular second amino acid to be used in a conservativesubstitution are known in the art. For example, when Thr; Asn; Gln; Cys;and Gly are available to be used in a conservative substitution for Ser,Cys may be eliminated from selection in order to avoid the formation ofundesirable cross-linkages and/or disulfide bonds. Likewise, Gly may beeliminated from selection, because it lacks an alkyl side chain. In thiscase, Thr may be selected, e.g., in order to retain the functionality ofa side chain hydroxyl group. The selection of the particular secondamino acid to be used in a conservative substitution is ultimately,however, within the discretion of the skilled practitioner.

The term “derivative” as used herein in relation to the amino acidsequence means chemical modification of a fusion protein of theinvention.

Transactivating protein: As used herein, the term “transactivatingprotein” (or “transactivator” or “transcriptional activator protein” or“transcriptional activator fusion protein”) refers to a polypeptide thatbinds to a nucleic acid element and initiates or enhances thetranscription of a polynucleotide (e.g., a gene of interest) that isoperably linked to the nucleic acid element. Transactivating proteinsthat are native to certain organisms include, for example and withoutlimitation, zinc finger DNA-binding proteins; UPA DNA-binding domain;GAL4; and TAL. Particular embodiments of the invention include syntheticfusion protein transactivators comprising at least one DNA-bindingdomain from a DNA-binding protein and an interaction motif from a planttransactivation domain.

Specific binding: As used herein with regard to polypeptides and proteindomains, the term “specific binding” refers to a sufficiently stronginteraction between the polypeptide or protein domain and its bindingpartner(s) (e.g., polypeptide(s) comprising a specific amino acidsequence, or nucleic acid(s) comprising a specific nucleotide sequence)such that stable and specific binding occurs with the bindingpartner(s), but not with other molecules that lack a specific amino acidsequence or specific nucleotide sequence that is recognized by thespecifically-binding polypeptide. Stable and specific binding may beascertained by techniques routine to those in the art; such as“pulldown” assays (e.g., GST pulldowns), yeast-2-hybrid assays,yeast-3-hybrid assays, ELISA, etc. Molecules that have the attribute of“specific binding” to each other may be said to “bind specifically” toeach other.

Transformation: As used herein, the term “transformation” refers to thetransfer of one or more nucleic acid molecule(s) into a cell. A cell is“transformed” by a nucleic acid molecule transferred into the cell whenthe nucleic acid molecule becomes stably replicated by the cell, eitherby incorporation of the nucleic acid molecule into the cellular genome,or by episomal replication. As used herein, the term “transformation”encompasses all techniques by which a nucleic acid molecule can beintroduced into such a cell. Examples include, but are not limited to:transfection with viral vectors; transformation with plasmid vectors;electroporation (Fromm et al. (1986) Nature 319:791-3); lipofection(Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7);microinjection (Mueller et al. (1978) Cell 15:579-85);Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad.Sci. USA 80:4803-7); direct DNA uptake; and microprojectile bombardment(Klein et al. (1987) Nature 327:70).

Transgene: An exogenous nucleic acid sequence. In some examples, atransgene may be a sequence that encodes a polypeptide comprising atleast one synthetic transcriptional activator fusion protein. In someexamples, a transgene may encode a synthetic transcriptional activatorfusion protein comprising at least one plant TAD and/or at least onevariant TAD. In some examples, a transgene may encode a gene of interest(e.g., a reporter gene; a gene conferring herbicide resistance; and agene contributing to an agriculturally important plant trait). In theseand other examples, a transgene may contain one or more regulatorysequences (e.g., a promoter) operably linked to a coding sequence of thetransgene. For the purposes of this disclosure, the term “transgenic,”when used to refer to an organism (e.g., a plant), refers to an organismthat comprises the exogenous nucleic acid sequence. In some examples,the organism comprising the exogenous nucleic acid sequence may be anorganism into which the nucleic acid sequence was introduced viamolecular transformation techniques. In other examples, the organismcomprising the exogenous nucleic acid sequence may be an organism intowhich the nucleic acid sequence was introduced by, for example,introgression or cross-pollination in a plant.

Vector: As used herein, the term “vector” refers to a nucleic acidmolecule as may be introduced into a cell, for example, to produce atransformed cell. A vector may include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.Examples of vectors include, but are not limited to: plasmids; cosmids;bacteriophages; and viruses that carry exogenous DNA into a cell. Avector may also include one or more genes, antisense molecules, and/orselectable marker genes and other genetic elements known in the art. Avector may transduce, transform, or infect a cell, thereby causing thecell to express the nucleic acid molecules and/or proteins encoded bythe vector. A vector optionally includes materials to aid in achievingentry of the nucleic acid molecule into the cell (e.g., a liposome,protein coating, etc.).

Unless specifically indicated or implied, the terms “a”, “an”, and “the”signify “at least one,” as used herein.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this disclosure belongs.Definitions of common terms in molecular biology can be found in, forexample, Lewin B., Genes V, Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and MeyersR. A. (ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

IV. Interaction Motifs' from Plant Transactivation Domains

This disclosure provides novel plant TADs and protein-proteininteraction motifs therefrom, as well as nucleic acids encoding thesame. TADs that were identified and isolated from the plant proteins,ERF2 (Arabidopsis thaliana); PTI4 (Solanum tuberosum); AtERF1 (A.thaliana); ORCA2 (Catharanthus roseus); DREB1A (A. thaliana); CBF1 (A.thaliana); and DOF1 (Zea mays), were used to identify plant TADinteraction motifs that may in some embodiments be “swapped” into aheterologous TAD, or used to produce a variant TAD interaction motif.The newly identified plant TADs, interaction motifs therein, and variantTADs thereof, may be used in particular embodiments (e.g., by inclusionin a synthetic transcriptional activator protein) to confer new anddesirable expression regulatory control to a gene of interest.

The TAD of VP 16 (V 16 TAD) has been characterized, and structuralregions of the foregoing novel plant TADs and interaction domains arereferred to by analogy to the corresponding structures in VP16 TAD. VP16TAD can be divided into two subdomains, and each subdomain is capable ofindependently and autonomously activating transcription when tethered toa DNA-binding domain. The VP16 subdomains are sometimes referred to asthe amino subdomain (or “VP16 transactivation subdomain I” or“VP16₄₁₂₋₄₅₆”) and the carboxyl subdomain (or “VP16 transactivationsubdomain II” or “VP16₄₅₆₋₄₉₀”). VP16 interacts through its interactiondomains with several target proteins involved in transcription,including the p62/Tfb1 subunit of transcription factor JIB (TFIIB).

The activity of VP16 depends not only on acidic residues, but also onhydrophobic and aromatic amino acids within the TAD. See, e.g., Cressand Triezenberg (1991) Science 251(4989):87-90. However, the acidic VP16TAD may be more tolerant to mutagenesis than many other polypeptidesequences, due to the lack of regular secondary structure in acidicTADs. Sigler (1988) Nature 333:210-2. The unstructured nature of theVP16 TAD subdomains may help the TAD mediate multiple protein/proteininteractions with different binding partners (Dyson and Wright (2002)Curr. Opin. Struct. Biol. 12(1):54-60), and the TAD subdomains may adopta more ordered structure (e.g., a short α-helix (Langlois et al. (2008),supra) when they are bound to target proteins than when free insolution. See, e.g., Garza et al. (2009) Life Sci. 84(7-8):189-93;Jonker et al. (2005) Biochemistry 44(3):827-39; Langlois et al. (2008),supra.

Some embodiments include a synthetic transcriptional activator proteincomprising a plant TAD interaction motif, wherein the plant TADinteraction motif is selected from the group consisting of SEQ ID NOs:10-16. Some exemplary synthetic transcriptional activator proteinscomprise a TAD that comprises a plant TAD interaction motif, and suchproteins may have a sequence including, for example and withoutlimitation, SEQ ID NOs: 2-8 and SEQ ID NOs: 100-106. Additionalexemplary TADs comprising a plant TAD interaction motif include thoseengineered by replacing a native TAD interaction motif sequencecomprised in a native transactivator TAD with one of SEQ ID NOs: 10-16.

Some embodiments of the invention take advantage of the surprisingdiscovery that variant TAD interaction motifs, comprising conservativeamino acid substitutions and/or substitutions with amino acids found inthe analogous position in a homologous interaction motif (e.g., from anortholog of the native protein comprising the reference TAD interactionmotif), may yield new and particular gene regulatory properties. Theseparticular properties may be desirable for the expression of apolynucleotide, wherein a certain level of expression is desired. Forexample, a variant TAD interaction motif may provide enhanced expressionover a native reference TAD motif that itself enhances expression, andthus may be desirable in protein synthesis and purification reactions,where maximized expression is often a goal. In other examples, a variantTAD interaction motif may provide less expression than a nativereference TAD motif where less than maximized expression is desired.

Thus, some embodiments include a synthetic transcriptional activatorprotein comprising a variant TAD interaction motif. In some examples, avariant TAD interaction motif may be a variant of one of SEQ ID NOs:10-16. Variant TAD interaction motifs include polypeptides having theamino acid sequence of a native TAD interaction sequence, but whereinone or more amino acids in the sequence have been changed to the aminoacid found at the corresponding position in a different, homologous TAD.Variant TAD interaction motifs also include polypeptides having theamino acid sequence of a native TAD interaction sequence, but wherein aconservative substitution has been made for one or more amino acids inthe sequence. A variant TAD interaction motif may be, for example andwithout limitation, at least 95% identical to a reference TADinteraction motif sequence (e.g., a sequence selected from SEQ ID NOs:10-16); at least 90% identical to the reference sequence; at least 85%identical to the reference sequence; at least 80% identical to thereference sequence; at least 75% identical to the reference sequence; atleast 70% identical to the reference sequence; at least 65% identical tothe reference sequence; at least 60% identical to the referencesequence; at least 55% identical to the reference sequence; at least 50%identical to the reference sequence; or less than 50% identical to thereference sequence.

Variant TAD interaction motifs include, for example and withoutlimitation, SEQ ID NOs: 17-22 (exemplary variant ERF2 TAD interactionmotifs); SEQ ID NOs: 23-28 (exemplary variant PTI4 TAD interactionmotifs); SEQ ID NOs: 29-34 (exemplary variant AtERF1 TAD interactionmotifs); SEQ ID NOs: 35-40 (exemplary variant ORCA2 TAD interactionmotifs); SEQ ID NOs: 41-46 (exemplary variant DREB1A TAD interactionmotifs); SEQ ID NOs: 47-52 (exemplary variant CBF1 TAD interactionmotifs); and SEQ ID NOs: 53-58 (exemplary variant DOF1 TAD interactionmotifs). Exemplary TADs comprising a variant TAD interaction motifinclude those engineered by replacing a TAD interaction motif sequencecomprised in a native transactivator TAD with a variant TAD selectedfrom the group consisting of SEQ ID NOs: 17-58. For example, exemplaryTADs comprising a variant TAD interaction motif include SEQ ID NOs:107-120.

Nucleic acids encoding any and all of the foregoing polypeptides areimmediately identifiable from the amino acid sequence of thepolypeptide. For example, a TAD or TAD interaction motif may be encodedby the native polynucleotide that is transcribed to generate an mRNAthat is subsequently translated into the amino acids of the TAD or TADinteraction motif. However, one of skill in the art will appreciatethat, due to the degeneracy of the genetic code, many other equivalentpolynucleotides exist that will encode an identical polypeptide. VariantTAD interaction motifs (e.g., SEQ ID NOs: 17-58) may be encoded bypolynucleotides that are readily determinable by reference to an RNAcodon table from the amino acid sequence of the particular variantdesired. In particular embodiments, it may be desirable for thenucleotide sequence of a polynucleotide encoding a TAD interaction motif(or variant thereof) to be assembled according to the codon usage of thehost cell, for example, so as to maximize or optimize expression of aprotein (e.g., a fusion protein) comprising the TAD interaction motif.

V. Fusion Protein Transcriptional Activators

This disclosure also provides synthetic transcriptional activator fusionproteins comprising a plant TAD interaction motif and/or a variant TADinteraction motif. In some embodiments, a synthetic transcriptionalactivator fusion protein further comprises at least one DNA-bindingdomain. Nucleic acids (e.g., DNA) encoding such synthetictranscriptional activator fusion proteins are also provided.

In some embodiments, a synthetic transcriptional activator fusionprotein comprises at least a first polypeptide that binds to DNA in asequence-specific manner (i.e., a “DNA-binding domain”). The firstDNA-binding domain polypeptide of the synthetic transcriptionalactivator fusion protein may be operatively linked to at least a secondpolypeptide comprising a plant TAD interaction motif or variant TADinteraction motif. In some examples, a synthetic transcriptionalactivator fusion protein may comprise additional polypeptides, such as aspacer sequence positioned between the first and second polypeptides inthe fusion protein; a leader peptide; a peptide that targets the fusionprotein to an organelle (e.g., the nucleus); polypeptides that arecleaved by a cellular enzyme; peptide tags; and other amino acidsequences that do not interfere with the function of the operativelylinked first and second polypeptides.

In some embodiments, the first and second polypeptides of a synthetictranscriptional activator fusion protein may be operatively linked bytheir expression from a single polynucleotide encoding the first andsecond polypeptides ligated to each other in-frame, so as to create achimeric gene encoding a fusion protein. Examples of polynucleotidesencoding a transcriptional activator fusion protein comprising aDNA-binding domain and a TAD interaction motif include, withoutlimitation, SEQ ID NOs: 79-93. In alternative embodiments, the first andsecond polypeptides of a synthetic transcriptional activator fusionprotein may be operatively linked by other means, such as bycross-linkage of independently expressed first and second polypeptides.

Plant TAD interaction motifs and variant TAD interaction motifs that maybe comprised within a synthetic transcriptional activator fusion proteininclude the TAD interaction motifs and variants thereof described inSection IV, supra. For example, a synthetic transcriptional activatorfusion protein may comprise a polypeptide selected from the groupconsisting of SEQ ID NOs: 10-58.

DNA-binding domains that may be comprised in a synthetic transcriptionalactivator fusion protein include zinc finger DNA-binding domains fromzinc finger proteins (e.g., a Z6 DNA-binding domain). Individual zincfinger DNA-binding domains can be designed to target and bind to a largerange of DNA sites. See, e.g., Wu et al. (2007) Cell. Mol. Life. Sci.64:2933-44. Canonical Cys₂His₂, as well as non-canonical Cys₃His zincfinger proteins, bind DNA by inserting an α-helix into the major grooveof the double helix. Recognition of DNA by zinc finger domains ismodular; each finger contacts primarily three consecutive base pairs inthe target, and a few key residues in the protein mediate recognition.By including multiple zinc finger DNA-binding domains in a synthetictranscriptional activator fusion protein, the DNA-binding specificity ofthe fusion protein may be further increased (and hence the specificityof any gene regulatory effects conferred thereby may also be increased).See, e.g., Urnov et al. (2005) Nature 435:646-51. Thus, one or more zincfinger DNA-binding domains may be engineered and utilized such that asynthetic transcriptional activator fusion protein introduced into ahost cell interacts with a DNA sequence that is unique within the genomeof the host cell.

In some examples, a synthetic transcriptional activator fusion proteincomprises a DNA-binding domain from GAL4, a modular transactivator inSaccharomyces cerevisiae, but which also operates as a transactivator inmany other organisms. See, e.g., Sadowski et al. (1988) Nature335:563-4. In this regulatory system, the expression of genes encodingenzymes of the galactose metabolic pathway in S. cerevisiae isstringently regulated by the available carbon source. Johnston (1987)Microbiol. Rev. 51:458-76. Transcriptional control of these metabolicenzymes is mediated by the interaction between the positive regulatoryprotein, GAL4, and a 17 bp symmetrical DNA sequence to which GAL4specifically binds (the UAS).

Native GAL4 consists of 881 amino acid residues, with a molecular weightof 99 kDa. GAL4 comprises functionally autonomous domains, the combinedactivities of which account for activity of GAL4 in vivo. Ma & Ptashne(1987) Cell 48:847-53); Brent & Ptashne (1985) Cell 43(3 Pt 2):729-36.The N-terminal 65 amino acids of GAL4 comprise the GAL4 DNA-bindingdomain. Keegan et al. (1986) Science 231:699-704; Johnston (1987) Nature328:353-5. Sequence-specific binding requires the presence of a divalentcation coordinated by 6 Cys residues present in the DNA binding domain.The coordinated cation-containing domain interacts with and recognizes aconserved CCG triplet at each end of the 17 bp UAS via direct contactswith the major groove of the DNA helix. Marmorstein et al. (1992) Nature356:408-14. The DNA-binding function of the protein positions C-terminaltranscriptional activating domains in the vicinity of the promoter, suchthat the activating domains can direct transcription.

Additional DNA-binding domains that may be comprised in a synthetictranscriptional activator fusion protein include, for example andwithout limitation, a binding sequence from a AVRBS3-inducible gene; aconsensus binding sequence from a AVRBS3-inducible gene or syntheticbinding sequence engineered therefrom (e.g., UPA DNA-binding domain; SEQID NO: 89); TAL; LexA (see, e.g., Brent & Ptashne (1985), supra); LacR(see, e.g., Labow et al. (1990) Mol. Cell. Biol. 10:3343-56; Baim et al.(1991) Proc. Natl. Acad. Sci. USA 88(12):5072-6); a steroid hormonereceptor (Ellliston et al. (1990) J. Biol. Chem. 265:11517-121); the Tetrepressor (U.S. Pat. No. 6,271,341) and a mutated Tet repressor thatbinds to a tet operator sequence in the presence, but not the absence,of tetracycline (Tc); and components of the regulatory system describedin Wang et al. (1994) Proc. Natl. Acad. Sci. USA 91(17):8180-4, whichutilizes a fusion of GAL4, a hormone receptor, and VP16.

In some examples, a synthetic transcriptional activator fusion proteincomprises more than one TAD interaction motif. For example and withoutlimitation, a synthetic transcriptional activator fusion protein maycomprise 2, 3, 4, or more TAD interaction domains. In some examples, asynthetic transcriptional activator fusion protein comprises more thanone DNA-binding domain. For example and without limitation, a synthetictranscriptional activator fusion protein may comprise 2, 3, 4, 5, 6, 7,8, 9, 10, or more DNA-binding domains.

VI. Nucleic Acid Molecules Comprising a Polynucleotide Encoding a FusionProtein Transcriptional Activator

In some embodiments, this disclosure provides a nucleic acid moleculecomprising at least one polynucleotide sequence encoding a plant TADinteraction motif, variant TAD interaction motif, plant TAD, or variantTAD. Such nucleic acid molecules may further comprise at least onepolynucleotide sequence encoding a DNA-binding domain. For example, anucleic acid in some embodiments comprises a first polynucleotidesequence encoding a plant TAD interaction motif, variant TAD interactionmotif, plant TAD, or variant TAD, fused in-frame to a secondpolynucleotide sequence encoding a DNA-binding domain, such that the twopolynucleotide sequences are transcribed as part of a single fusionprotein.

In nucleic acid molecules provided in some embodiments of the invention,the last codon of a first polynucleotide sequence encoding a plant TADinteraction motif, variant TAD interaction motif, plant TAD, or variantTAD, and the first codon of a second polynucleotide sequence encoding aDNA-binding domain may be separated by any number of nucleotidetriplets, e.g., without coding for an intron or a “STOP.” Likewise, thelast codon of a nucleotide sequence encoding a first polynucleotidesequence encoding a DNA-binding domain, and the first codon of a secondpolynucleotide sequence encoding a plant TAD interaction motif, variantTAD interaction motif, plant TAD, or variant TAD, may be separated byany number of nucleotide triplets. In these and further embodiments, thelast codon of the last (i.e., most 3′ in the nucleic acid sequence) ofthe first polynucleotide sequence encoding a plant TAD interactionmotif, variant TAD interaction motif, plant TAD, or variant TAD, and thesecond polynucleotide sequence encoding a DNA-binding domain, may befused in phase-register with the first codon of a further polynucleotidecoding sequence directly contiguous thereto, or separated therefrom byno more than a short peptide sequence, such as that encoded by asynthetic nucleotide linker (e.g., a nucleotide linker that may havebeen used to achieve the fusion). Examples of such furtherpolynucleotide sequences include, for example and without limitation,tags, targeting peptides, and enzymatic cleavage sites. Likewise, thefirst codon of the most 5′ (in the nucleic acid sequence) of the firstand second polynucleotide sequences may be fused in phase-register withthe last codon of a further polynucleotide coding sequence directlycontiguous thereto, or separated therefrom by no more than a shortpeptide sequence.

A sequence separating a polynucleotide sequence encoding a plant TADinteraction motif, variant TAD interaction motif, plant TAD, or variantTAD, and a polynucleotide sequence encoding a DNA-binding domain may,for example, consist of any sequence, such that the amino acid sequenceencoded is not likely to significantly alter the translation of thefusion protein. Due to the autonomous nature of the TAD interactiondomains (and variants thereof) disclosed herein and known DNA-bindingdomains, intervening sequences will not in examples interfere with therespective functions of these structures.

Some embodiments of the invention also include a nucleic acid moleculecomprising a polynucleotide sequence encoding a plant TAD interactionmotif, variant TAD interaction motif, plant TAD, or variant TAD, whereinthe nucleic acid molecule does not comprise a polynucleotide sequenceencoding a DNA-binding domain. Such nucleic acid molecules may beuseful, for example, in facilitating manipulation of the TAD interactionmotif-encoding sequence in molecular biology techniques. For example, insome embodiments, a TAD interaction motif-encoding sequence may beintroduced into a suitable vector for sub-cloning of the sequence intoan expression vector, or a TAD interaction motif-encoding sequence maybe introduced into a nucleic acid molecule that facilitates theproduction of a further nucleic acid molecule comprising the TADinteraction motif-encoding sequence operably linked to a nucleotidesequence of interest.

All of the nucleotide sequences that encode, for example, a fusionprotein comprising at least one particular plant TAD interaction motif,variant TAD interaction motif, plant TAD, or variant TAD, and furthercomprising at least one particular DNA-binding domain, will beimmediately recognizable by those of skill in the art. The degeneracy ofthe genetic code provides a finite number of coding sequences for aparticular amino acid sequence. The selection of a particular sequenceto encode a fusion protein according to embodiments of the invention iswithin the discretion of the practitioner. Different coding sequencesmay be desirable in different applications.

In some embodiments, it may be desirable to modify the nucleotides of apolynucleotide sequence encoding a plant TAD interaction motif, variantTAD interaction motif, plant TAD, or variant TAD (and/or nucleotides ofa DNA-binding domain-encoding sequence), for example, to enhanceexpression of the polynucleotide sequence in a particular host. Thegenetic code is redundant with 64 possible codons, but most organismpreferentially use a subset of these codons. The codons that areutilized most often in a species are called optimal codons, and thosenot utilized very often are classified as rare or low-usage codons.Zhang et al. (1991) Gene 105:61-72. Codons may be substituted to reflectthe preferred codon usage of a particular host in a process sometimesreferred to as “codon optimization.” Optimized coding sequencescontaining codons preferred by a particular prokaryotic or eukaryotichost may be prepared by, for example, to increase the rate oftranslation or to produce recombinant RNA transcripts having desirableproperties (e.g., a longer half-life, as compared with transcriptsproduced from a non-optimized sequence).

VII. Expression of a Fusion Protein Transcriptional Activator

In some embodiments, at least one fusion protein-encoding nucleic acidmolecule(s) comprising at least one polynucleotide sequence encoding aplant TAD interaction motif (or variant TAD interaction motif, plantTAD, or variant TAD), and at least one polynucleotide sequence encodinga DNA-binding domain, may be introduced into a cell, tissue, or organismfor expression of the fusion protein therein.

In some embodiments, such a nucleic acid molecule may, for example, be avector system including, for example and without limitation, a linearplasmid, and a closed circular plasmid. In particular examples, thevector may be an expression vector. Nucleic acid sequences according toparticular embodiments may, for example, be inserted into a vector, suchthat the nucleic acid sequence is operably linked to one or moreregulatory sequences. Many vectors are available for this purpose, andselection of the particular vector may depend, for example, on the sizeof the nucleic acid to be inserted into the vector, the particular hostcell to be transformed with the vector, and/or the amount of the fusionprotein that is desired to be expressed. A vector typically containsvarious components, the identity of which depend on a function of thevector (e.g., amplification of DNA and expression of DNA), and theparticular host cell(s) with which the vector is compatible.

Some embodiments may include a plant transformation vector thatcomprises a nucleotide sequence comprising at least one regulatorysequence operably linked to one or more nucleotide sequence(s) encodinga fusion protein comprising at least one plant TAD interaction motif,variant TAD interaction motif, plant TAD, or variant TAD, operativelylinked to at least one DNA-binding domain. The one or more nucleotidesequence(s) may be expressed, under the control of the regulatorysequence(s), in a plant cell, tissue, or organism to produce the fusionprotein.

In some embodiments, a regulatory sequence operably linked to one ormore nucleotide sequence(s) encoding a fusion protein comprising atleast one plant TAD interaction motif, variant TAD interaction motif,plant TAD, or variant TAD, operatively linked to at least oneDNA-binding domain, may be a promoter sequence that functions in a hostcell, such as a bacterial cell, wherein the nucleic acid molecule is tobe amplified, or a plant cell wherein the nucleic acid molecule is to beexpressed.

Promoters suitable for use in nucleic acid molecules according to someembodiments include those that are inducible, viral, synthetic, orconstitutive, all of which are well known in the art. Non-limitingexamples of promoters that may be useful in embodiments of the inventionare provided by: U.S. Pat. Nos. 6,437,217 (maize RS81 promoter);5,641,876 (rice actin promoter); 6,426,446 (maize RS324 promoter);6,429,362 (maize PR-1 promoter); 6,232,526 (maize A3 promoter);6,177,611 (constitutive maize promoters); 5,322,938, 5,352,605,5,359,142, and 5,530,196 (35S promoter); 6,433,252 (maize L3 oleosinpromoter); 6,429,357 (rice actin 2 promoter, and rice actin 2 intron);6,294,714 (light-inducible promoters); 6,140,078 (salt-induciblepromoters); 6,252,138 (pathogen-inducible promoters); 6,175,060(phosphorous deficiency-inducible promoters); 6,388,170 (bidirectionalpromoters); 6,635,806 (gamma-coixin promoter); and U.S. patentapplication Ser. No. 09/757,089 (maize chloroplast aldolase promoter).

Additional exemplary promoters include the nopaline synthase (NOS)promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-9);the octopine synthase (OCS) promoter (which is carried on tumor-inducingplasmids of Agrobacterium tumefaciens); the caulimovirus promoters suchas the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al.(1987) Plant Mol. Biol. 9:315-24); the CaMV 35S promoter (Odell et al.(1985) Nature 313:810-2; the figwort mosaic virus 35S-promoter (Walkeret al. (1987) Proc. Natl. Acad. Sci. USA 84(19):6624-8); the sucrosesynthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA87:4144-8); the R gene complex promoter (Chandler et al. (1989) PlantCell 1:1175-83); the chlorophyll a/b binding protein gene promoter;CaMV35S (U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196);FMV35S (U.S. Pat. Nos. 6,051,753, and 5,378,619); a PC1SV promoter (U.S.Pat. No. 5,850,019); the SCP1 promoter (U.S. Pat. No. 6,677,503); andAGRtu.nos promoters (GenBank Accession No. V00087; Depicker et al.(1982) J. Mol. Appl. Genet. 1:561-73; Bevan et al. (1983) Nature304:184-7).

In particular embodiments, nucleic acid molecules of the invention maycomprise a tissue-specific promoter. A tissue-specific promoter is anucleotide sequence that directs a higher level of transcription of anoperably linked nucleotide sequence in the tissue for which the promoteris specific, relative to the other tissues of the organism. Examples oftissue-specific promoters include, without limitation: tapetum-specificpromoters; anther-specific promoters; pollen-specific promoters (See,e.g., U.S. Pat. No. 7,141,424, and International PCT Publication No. WO99/042587); ovule-specific promoters; (See, e.g., U.S. PatentApplication No. 2001/047525 A1); fruit-specific promoters (See, e.g.,U.S. Pat. Nos. 4,943,674, and 5,753,475); and seed-specific promoters(See, e.g., U.S. Pat. Nos. 5,420,034, and 5,608,152). In someembodiments, a developmental stage-specific promoter (e.g., a promoteractive at a later stage in development) may be used in a composition ormethod of the invention.

Additional regulatory sequences that may in some embodiments be operablylinked to a nucleic acid molecule include 5′ UTRs located between apromoter sequence and a coding sequence that function as a translationleader sequence. The translation leader sequence is present in thefully-processed mRNA, and it may affect processing of the primarytranscript, and/or RNA stability. Examples of translation leadersequences include maize and petunia heat shock protein leaders (U.S.Pat. No. 5,362,865), plant virus coat protein leaders, plant rubiscoleaders, and others. See, e.g., Turner and Foster (1995) MolecularBiotech. 3(3):225-36. Non-limiting examples of 5′ UTRs are provided by:GmHsp (U.S. Pat. No. 5,659,122); PhDnaK (U.S. Pat. No. 5,362,865);AtAnt1; TEV (Carrington and Freed (1990) J. Virol. 64:1590-7); andAGRtunos (GenBank Accession No. V00087; and Bevan et al. (1983), supra).

Additional regulatory sequences that may in some embodiments be operablylinked to a nucleic acid molecule also include 3′ non-translatedsequences, 3′ transcription termination regions, or poly-adenylationregions. These are genetic elements located downstream of a nucleotidesequence, and include polynucleotides that provide polyadenylationsignal, and/or other regulatory signals capable of affectingtranscription or mRNA processing. The polyadenylation signal functionsin plants to cause the addition of polyadenylate nucleotides to the 3′end of the mRNA precursor. The polyadenylation sequence can be derivedfrom a variety of plant genes, or from T-DNA genes. A non-limitingexample of a 3′ transcription termination region is the nopalinesynthase 3′ region (nos 3′; Fraley et al. (1983) Proc. Natl. Acad. Sci.USA 80:4803-7). An example of the use of different 3′ nontranslatedregions is provided in Ingelbrecht et al. (1989) Plant Cell 1:671-80.Non-limiting examples of polyadenylation signals include one from aPisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. (1984) EMBO J.3:1671-9) and AGRtu.nos (GenBank Accession No. E01312).

Additional information regarding regulatory sequences that may be usefulin particular embodiments is described, for example, in Goeddel (1990)“Gene Expression Technology,” Methods Enzymol. 185, Academic Press, SanDiego, Calif.

A recombinant nucleic acid molecule or vector of the present inventionmay comprise a selectable marker that confers a selectable phenotype ona transformed cell, such as a plant cell. Selectable markers may also beused to select for plants or plant cells that comprise a nucleic acidmolecule of the invention. The marker may encode biocide resistance,antibiotic resistance (e.g., kanamycin, Geneticin (G418), bleomycin, andhygromycin), or herbicide resistance (e.g., glyphosate). Examples ofselectable markers include, but are not limited to: a neo gene thatconfers kanamycin resistance and can be selected for using, e.g.,kanamycin and G418; a bar gene that confers bialaphos resistance; amutant EPSP synthase gene that confers glyphosate resistance; anitrilase gene that confers resistance to bromoxynil; a mutantacetolactate synthase gene (ALS) that confers imidazolinone orsulfonylurea resistance; and a methotrexate-resistant DHFR gene.Multiple selectable markers are available that confer resistance tochemical agents including, for example and without limitation,ampicillin; bleomycin; chloramphenicol; gentamycin; hygromycin;kanamycin; lincomycin; methotrexate; phosphinothricin; puromycin;spectinomycin; rifampicin; streptomycin; and tetracycline. Examples ofsuch selectable markers are illustrated in, e.g., U.S. Pat. Nos.5,550,318; 5,633,435; 5,780,708 and 6,118,047.

A nucleic acid molecule or vector of the present invention may also oralternatively include a screenable marker. Screenable markers may beused to monitor expression. Exemplary screenable markers include aβ-glucuronidase or uidA gene (GUS) which encodes an enzyme for whichvarious chromogenic substrates are known (Jefferson et al. (1987) PlantMol. Biol. Rep. 5:387-405); an R-locus gene, which encodes a productthat regulates the production of anthocyanin pigments (red color) inplant tissues (Dellaporta et al. (1988) “Molecular cloning of the maizeR-nj allele by transposon tagging with Ac.” In 18th Stadler GeneticsSymposium, P. Gustafson and R. Appels, eds., Plenum, NY (pp. 263-82); aβ-lactamase gene (Sutcliffe et al. (1978) Proc. Natl. Acad. Sci. USA75:3737-41); a gene which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene (Ow et al. (1986) Science 234:856-9);a xylE gene that encodes a catechol dioxygenase that convertschromogenic catechols (Zukowski et al. (1983) Gene 46(2-3):247-55); anamylase gene (Ikatu et al. (1990) Bio/Technol. 8:241-2); a tyrosinasegene which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone, which in turn condenses to melanin (Katz et al. (1983) J.Gen. Microbiol. 129:2703-14); and an α-galactosidase.

Suitable methods for transformation of host cells include any method bywhich DNA can be introduced into a cell, for example and withoutlimitation: by transformation of protoplasts (See, e.g., U.S. Pat. No.5,508,184); by desiccation/inhibition-mediated DNA uptake (See, e.g.,Potrykus et al. (1985) Mol. Gen. Genet. 199:183-8); by electroporation(See, e.g., U.S. Pat. No. 5,384,253); by agitation with silicon carbidefibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765); byAgrobacterium-mediated transformation (See, e.g., U.S. Pat. Nos.5,563,055, 5,591,616, 5,693,512, 5,824,877, 5,981,840, and 6,384,301);and by acceleration of DNA-coated particles (See, e.g., U.S. Pat. Nos.5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865).Through the application of techniques such as these, the cells ofvirtually any species may be stably transformed. In some embodiments,transforming DNA is integrated into the genome of the host cell. In thecase of multicellular species, transgenic cells may be regenerated intoa transgenic organism. Any of these techniques may be used to produce atransgenic plant, for example, comprising one or more nucleic acidsequences of the invention in the genome of the transgenic plant.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria that genetically transform plant cells. The T_(i) andR_(i) plasmids of A. tumefaciens and A. rhizogenes, respectively, carrygenes responsible for genetic transformation of the plant. The T_(i)(tumor-inducing)-plasmids contain a large segment, known as T-DNA, whichis transferred to transformed plants. Another segment of the T_(i)plasmid, the vir region, is responsible for T-DNA transfer. The T-DNAregion is bordered by left-hand and right-hand borders that are eachcomposed of terminal repeated nucleotide sequences. In some modifiedbinary vectors, the tumor-inducing genes have been deleted, and thefunctions of the vir region are utilized to transfer foreign DNAbordered by the T-DNA border sequences. The T-region may also contain,for example, a selectable marker for efficient recovery of transgenicplants and cells, and a multiple cloning site for inserting sequencesfor transfer such as a nucleic acid encoding a fusion protein of theinvention.

Thus, in some embodiments, a plant transformation vector is derived froma T_(i) plasmid of A. tumefaciens (See, e.g., U.S. Pat. Nos. 4,536,475,4,693,977, 4,886,937, and 5,501,967; and European Patent EP 0 122 791)or a R_(i) plasmid of A. rhizogenes. Additional plant transformationvectors include, for example and without limitation, those described byHerrera-Estrella et al. (1983) Nature 303:209-13; Bevan et al. (1983),supra; Klee et al. (1985) Bio/Technol. 3:637-42; and in European PatentEP 0 120 516, and those derived from any of the foregoing. Otherbacteria, such as Sinorhizobium, Rhizobium, and Mesorhizobium, thatnaturally interact with plants can be modified to mediate gene transferto a number of diverse plants. These plant-associated symbiotic bacteriacan be made competent for gene transfer by acquisition of both adisarmed T_(i) plasmid and a suitable binary vector.

After providing exogenous DNA to recipient cells, transformed cells aregenerally identified for further culturing and plant regeneration. Inorder to improve the ability to identify transformed cells, one maydesire to employ a selectable or screenable marker gene, as previouslyset forth, with the vector used to generate the transformant. In thecase where a selectable marker is used, transformed cells are identifiedwithin the potentially transformed cell population by exposing the cellsto a selective agent or agents. In the case where a screenable marker isused, cells may be screened for the desired marker gene trait.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In some embodiments, any suitableplant tissue culture media (e.g., MS and N6 media) may be modified byincluding further substances, such as growth regulators. Tissue may bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration (e.g., at least 2 weeks), then transferredto media conducive to shoot formation. Cultures are transferredperiodically until sufficient shoot formation has occurred. Once shootsare formed, they are transferred to media conducive to root formation.Once sufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

To confirm the presence of a nucleic acid molecule of interest (forexample, a nucleotide sequence encoding a polypeptide comprising atleast one fusion protein of the invention) in a regenerating plant, avariety of assays may be performed. Such assays include, for example:molecular biological assays, such as Southern and Northern blotting,PCR, and nucleic acid sequencing; biochemical assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAand/or Western blots) or by enzymatic function; plant part assays, suchas leaf or root assays; and analysis of the phenotype of the wholeregenerated plant.

Integration events may be analyzed, for example, by PCR amplificationusing, e.g., oligonucleotide primers that are specific for a nucleotidesequence of interest. PCR genotyping is understood to include, but notbe limited to, polymerase-chain reaction (PCR) amplification of genomicDNA derived from isolated host plant tissue predicted to contain anucleic acid molecule of interest integrated into the genome, followedby standard cloning and sequence analysis of PCR amplification products.Methods of PCR genotyping have been well described (see, e.g., Rios, G.et al. (2002) Plant J. 32:243-53), and may be applied to genomic DNAderived from any plant species or tissue type, including cell cultures.

A transgenic plant formed using Agrobacterium-dependent transformationmethods typically contains a single recombinant DNA sequence insertedinto one chromosome. The single recombinant DNA sequence is referred toas a “transgenic event” or “integration event.” Such transgenic plantsare heterozygous for the inserted DNA sequence. In some embodiments, atransgenic plant homozygous with respect to a transgene may be obtainedby sexually mating (selfing) an independent segregant transgenic plantthat contains a single exogenous gene sequence to itself, for example,an F₀ plant, to produce F₁ seed. One fourth of the F₁ seed produced willbe homozygous with respect to the transgene. Germinating F₁ seed resultsin plants that can be tested for heterozygosity, typically using a SNPassay or a thermal amplification assay that allows for the distinctionbetween heterozygotes and homozygotes (i.e., a zygosity assay).

In particular embodiments, copies of at least one synthetictranscriptional activator fusion protein comprising at least one plantTAD interaction motif (and/or variant TAD interaction motif) and atleast one DNA-binding domain are produced in a cell, into which has beenintroduced at least one nucleic acid molecule(s) comprising a nucleotidesequence encoding the at least one synthetic transcriptional activatorfusion protein. Each synthetic transcriptional activator fusion proteinmay be expressed from multiple nucleic acid sequences introduced indifferent transformation events, or from a single nucleic acid sequenceintroduced in a single transformation event. In some embodiments, aplurality of such fusion proteins may be expressed under the control ofa single promoter. In other embodiments, a plurality of such fusionproteins may be expressed under the control of multiple promoters.

In addition to direct transformation of a plant or plant cell with anucleic acid molecule of the invention, transgenic plants may beprepared in some embodiments by crossing a first plant having at leastone transgenic event with a second plant lacking such an event. Forexample, a nucleic acid molecule comprising a nucleotide sequenceencoding a synthetic transcriptional activator fusion protein comprisingat least one plant TAD interaction motif (and/or variant TAD interactionmotif) and at least one DNA-binding domain may be introduced into afirst plant line that is amenable to transformation, to produce atransgenic plant, which transgenic plant may be crossed with a secondplant line to introgress the nucleotide sequence that encodes thesynthetic transcriptional activator fusion protein into the second plantline.

VIII. Plant Materials Comprising a Fusion Protein TranscriptionalActivator

In some embodiments, a plant is provided, wherein the plant comprises aplant cell comprising a nucleotide sequence encoding a synthetictranscriptional activator fusion protein comprising at least one plantTAD interaction motif (and/or variant TAD interaction motif) and atleast one DNA-binding domain. In particular embodiments, such a plantmay be produced by transformation of a plant tissue or plant cell, andregeneration of a whole plant. In further embodiments, such a plant maybe obtained through introgression of a nucleic acid comprising anucleotide sequence encoding a synthetic transcriptional activatorfusion protein into a germplasm. Plant materials comprising such a plantcell are also provided. Such a plant material may be obtained from aplant comprising the plant cell.

A transgenic plant or plant material comprising a nucleotide sequenceencoding a synthetic transcriptional activator fusion protein comprisingat least one plant TAD interaction motif (and/or variant TAD interactionmotif) and at least one DNA-binding domain may in some embodimentsexhibit one or more of the following characteristics: expression of thefusion protein in a cell of the plant; expression of the fusion proteinin a plastid of a cell of the plant; expression of the fusion protein inthe nucleus of a cell of the plant; localization of the fusion proteinin a cell of the plant; integration of the nucleotide sequence in thegenome of a cell of the plant; presence of the nucleotide sequence inextra-chromosomal DNA of a cell of the plant; and/or the presence of anRNA transcript corresponding to the nucleotide sequence in a cell of theplant. Such a plant may additionally have one or more desirable traitsother than expression of the encoded fusion protein. Such traits mayinclude those resulting from the expression of an endogenous ortransgenic nucleotide sequence, the expression of which is regulated bythe fusion protein in a cell of the plant, for example and withoutlimitation: resistance to insects, other pests, and disease-causingagents; tolerances to herbicides; enhanced stability, yield, orshelf-life; environmental tolerances; pharmaceutical production;industrial product production; and nutritional enhancements.

A transgenic plant according to the invention may be any plant capableof being transformed with a nucleic acid molecule of the invention.Accordingly, the plant may be a dicot or monocot. Non-limiting examplesof dicotyledonous plants usable in the present methods includeArabidopsis, alfalfa, beans, broccoli, cabbage, canola, carrot,cauliflower, celery, Chinese cabbage, cotton, cucumber, eggplant,lettuce, melon, pea, pepper, peanut, potato, pumpkin, radish, rapeseed,spinach, soybean, squash, sugarbeet, sunflower, tobacco, tomato, andwatermelon. Non-limiting examples of monocotyledonous plants usable inthe present methods include corn, onion, rice, sorghum, wheat, rye,millet, sugarcane, oat, triticale, switchgrass, and turfgrass.Transgenic plants according to the invention may be used or cultivatedin any manner.

Some embodiments also provide commodity products containing one or morenucleotide sequences encoding a synthetic transcriptional activatorfusion protein comprising at least one plant TAD interaction motif(and/or variant TAD interaction motif) and at least one DNA-bindingdomain; for example, a commodity product produced from a recombinantplant or seed containing one or more of such nucleotide sequences.Commodity products containing one or more nucleotide sequences encodinga synthetic transcriptional activator fusion protein comprising at leastone plant TAD interaction motif (and/or variant TAD interaction motif)and at least one DNA-binding domain include, for example and withoutlimitation: food products, meals, oils, or crushed or whole grains orseeds of a plant comprising one or more nucleotide sequences encodingsuch a synthetic transcriptional activator fusion protein. The detectionof one or more nucleotide sequences encoding a synthetic transcriptionalactivator fusion protein of the invention in one or more commodity orcommodity products is de facto evidence that the commodity or commodityproduct was at least in part produced from a plant comprising one ormore nucleotide sequences encoding a synthetic transcriptional activatorfusion protein of the invention. In particular embodiments, a commodityproduct of the invention comprise a detectable amount of a nucleic acidsequence encoding a synthetic transcriptional activator fusion proteincomprising at least one plant TAD interaction motif (and/or variant TADinteraction motif) and at least one DNA-binding domain. In someembodiments, such commodity products may be produced, for example, byobtaining transgenic plants and preparing food or feed from them.

In some embodiments, a transgenic plant or seed comprising a transgenecomprising a nucleotide sequence encoding a synthetic transcriptionalactivator fusion protein of the invention also may comprise at least oneother transgenic event in its genome, including without limitation: atransgenic event from which is transcribed an RNAi molecule; a geneencoding an insecticidal protein (e.g., a Bacillus thuringiensisinsecticidal protein); an herbicide tolerance gene (e.g., a geneproviding tolerance to glyphosate); and a gene contributing to adesirable phenotype in the transgenic plant (e.g., increased yield,altered fatty acid metabolism, or restoration of cytoplasmic malesterility).

In some embodiments, a transgenic plant or seed comprising a transgenecomprising a nucleotide sequence encoding a synthetic transcriptionalactivator fusion protein of the invention may comprise an endogenous ornative gene target within the genome of the transgenic plant, includingwithout limitation: an endogenous gene target for an altered fatty acidmetabolism trait, an endogenous gene target for a drought tolerancetrait, an endogenous gene target for a nitrogen use efficiency trait, orany other endogenous gene target contributing to a desirable phenotypein the transgenic plant (e.g., increased yield, or restoration ofcytoplasmic male sterility). The endogenous or native gene target may beoperably linked to a nucleotide sequence to which the synthetictranscriptional activator fusion protein binds specifically, therebyaffecting transcription of the target gene.

IX. Regulation of Expression by a Fusion Protein TranscriptionalActivator

In some embodiments, a synthetic transcriptional activator fusionprotein comprising at least one plant TAD interaction motif (and/orvariant TAD interaction motif) and at least one DNA-binding domain maybe used to increase (e.g., initiate) expression of a nucleotide sequenceof interest (e.g., a gene of interest) in a cell. The nucleotidesequence of interest may in some embodiments be endogenous to the genomeof the cell. In other embodiments, at least one exogenous nucleic acidmolecule(s) comprising the nucleotide sequence of interest has beenintroduced into the cell. Generally, a second nucleotide sequenceoperably linked to the nucleotide sequence of interest will berecognized by the DNA-binding domain of the fusion protein, such thatstable and specific binding between the second nucleotide sequence andthe fusion protein can occur. In some examples, the at least one nucleicacid molecule(s) comprising the nucleotide sequence of interest furthercomprise such a second nucleotide sequence. In some examples, the atleast one nucleic acid molecule(s) comprising the nucleotide sequence ofinterest are introduced into the host cell, such that the nucleotidesequence of interest is operably linked to a second nucleotide sequencethat is endogenous to the host cell. For example, a nucleic acidmolecule comprising the nucleotide sequence of interest may facilitatehomologous recombination that inserts the nucleotide sequence ofinterest into the host cell's genome, such that the nucleotide sequenceof interest is operably linked to an endogenous sequence that isrecognized by a DNA-binding domain. In some examples, the at least onenucleic acid molecule(s) comprising the nucleotide sequence of interestis endogenous or native within the host cell, such that the nucleotidesequence of interest is operably linked to a second nucleotide sequencethat is endogenous to the host cell.

Multiple nucleotide sequence(s) of interest that are introduced indifferent transformation events may be expressed under the regulatorycontrol of a single fusion protein in some examples. In other examples,a single nucleotide sequence of interest (e.g., a single integrationevent) is regulated and expressed. In some embodiments, a plurality ofnucleotide sequences of interest may be regulated by the binding of afusion protein of the invention to a single nucleic acid binding site;for example, the plurality of nucleotide sequences of interest may allbe operably linked to the same second nucleotide sequence to which theDNA-binding domain of the fusion protein specifically binds. Thenucleotide sequences of interest comprising such a plurality are notnecessarily the same in certain examples. Thus, multiple different geneproducts may be expressed under the regulatory control of a singlefusion protein.

In particular embodiments, the expression product of a nucleotidesequence of interest that is under the regulatory control of a fusionprotein of the invention may be a marker gene product; for example andwithout limitation, a fluorescent molecule. Quantitative and qualitativeobservations regarding the expression of such an expression product mayprovide a system to evaluate the particular regulatory properties of aparticular TAD interaction motif or TAD interaction motif variant.

Any expression product (e.g., protein, precursor protein, and inhibitoryRNA molecule) may be expressed under the regulatory control of asynthetic transcriptional activator fusion protein comprising at leastone plant TAD interaction motif (and/or variant TAD interaction motif)and at least one DNA-binding domain. In particular examples, anexpression product under the regulatory control of a synthetictranscriptional activator fusion protein may be, without limitation, anendogenous or native polypeptide that is normally expressed in the hostcell into which a nucleic acid encoding the fusion protein isintroduced. In other examples, an expression product under theregulatory control of a synthetic transcriptional activator fusionprotein may be a heterologous polypeptide that is not normally expressedin the host cell. For example and without limitation, an expressionproduct under the regulatory control of a synthetic transcriptionalactivator fusion protein may be a polypeptide involved in herbicideresistance, virus resistance, bacterial pathogen resistance, insectresistance, nematode resistance, or fungal resistance. See, e.g., U.S.Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. Anexpression product under the regulatory control of a synthetictranscriptional activator fusion protein may alternatively be, forexample and without limitation, a polypeptide involved in plant vigor oryield (including polypeptides involved in tolerance for extremetemperatures, soil conditions, light levels, water levels, and chemicalenvironment), or a polypeptide that may be used as a marker to identifya plant comprising a trait of interest (e.g., a selectable marker geneproduct, and a polypeptide involved in seed color).

Non-limiting examples of polypeptides that may be under the regulatorycontrol of a synthetic transcriptional activator fusion proteincomprising at least one plant TAD interaction motif (and/or variant TADinteraction motif) and at least one DNA-binding domain in someembodiments of the invention include: acetolactase synthase (ALS),mutated ALS, and precursors of ALS (see, e.g., U.S. Pat. No. 5,013,659);EPSPS (see, e.g., U.S. Pat. Nos. 4,971,908 and 6,225,114), such as a CP4EPSPS or a class III EPSPS; enzymes that modify a physiological processthat occurs in a plastid, including for example and without limitation,photosynthesis, and synthesis of fatty acids, amino acids, oils,arotenoids, terpenoids, and starch. Other non-limiting examples ofpolypeptides that may be under the regulatory control of a synthetictranscriptional activator fusion protein in particular embodimentsinclude: zeaxanthin epoxidase, choline monooxygenase, ferrochelatase,omega-3 fatty acid desaturase, glutamine synthetase, starch modifyingenzymes, polypeptides involved in synthesis of essential amino acids,provitamin A, hormones, Bt toxin, and proteins. Nucleotide sequencesencoding the aforementioned peptides are available in the art, and suchnucleotide sequences may be operably linked to a specific binding sitefor a DNA-binding domain to be expressed under the regulatory control ofa synthetic transcriptional activator fusion protein comprising at leastone plant TAD interaction motif (and/or variant TAD interaction motif)and at least one DNA-binding domain that specifically binds to theoperably linked site.

Furthermore, a variant nucleotide sequence of interest encoding any ofthe aforementioned polypeptides to be placed under regulatory controlmay be identified by those of skill in the art (for example, by cloningof genes with high homology to other genes encoding the particularpolypeptide, or by in silico sequence generation in view of DNA codondegeneracy). Such variants may be desirable in particular embodiments,for example, to conform to the preferred codon usage of a host organism.Once such a variant nucleotide sequence of interest has been identified,a nucleic acid molecule to provide regulatory control of the sequence bya synthetic transcriptional activator polypeptide according to theinvention may be designed, for example by operably linking the variantnucleotide sequence of interest in the nucleic acid molecule to a knownbinding site for the DNA-binding domain comprised within the particularsynthetic transcriptional activator fusion protein to be used. Inembodiments described herein, a surprising increase in the expression ofsuch a variant nucleotide sequence of interest may be observed (e.g., ina host plant cell) when the nucleic acid molecule and one of theparticular synthetic transcriptional activator fusion proteins describedherein are present in a host cell at the same time.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to theextent they are not inconsistent with the explicit details of thisdisclosure, and are so incorporated to the same extent as if eachreference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. The examples should not be construed tolimit the disclosure to the particular features or embodimentsexemplified.

EXAMPLES Example 1 Identification of Plant Transactivation InteractionMotifs

Seven proteins were identified as homologous to the VP16 transactivationdomain (SEQ ID NO: 1): PTI4 (GenBank Accession No. ACF57857.1), ERF2(GenBank Accession No. NP_(—)199533.1), AtERF1(GenBank Accession No.NP_(—)567530.4), ORCA2 (GenBank Accession No. CAB93940.1), DREB1A(GenBank Accession No. NP_(—)567720.1), CBF1 (GenBank Accession No. NP567721.1), and DOF1 (GenBank Accession No. NP_(—)001105709.1). The aminoacid sequence of the VP16 transactivation domain (SEQ ID NO: 1) sharedsequence similarity with regions of these putative plant transcriptionactivators, and the VP16 sequence was used to locate a transactivationdomain within each activator. The identified transactivation domains forthese plant activator proteins are: SEQ ID NO: 2 (PTI4), SEQ ID NO: 3(ERF2), SEQ ID NO: 4 (AtERF1), SEQ ID NO: 5 (ORCA2), SEQ ID NO: 6(DREB1A), SEQ ID NO: 7 (CBF1), and SEQ ID NO: 8 (DOF1). Next, theinteraction motif of the VP16 transactivation subdomain II (FIG. 1; SEQID NO: 9) was used to locate an interaction motif from the planttransactivation domains. FIG. 2 shows an alignment of VP16 with theplant transactivation domains, wherein the novel interaction motifs arehighlighted.

Example 2 Modification of the Identified Interaction Motifs of PlantTransactivation Domains

The interaction motifs of the identified plant transactivation domainswere modified. New variants of the interaction motifs that contained theamino acid contact residues of the interaction motif of subdomain II ofthe VP16 transactivation domain were produced. Langlois et al. (2008) J.Am. Chem. Soc. 130:10596. Six amino acid contact residues of the VP16transactivation domain are proposed to directly interact with Tfb1, asubunit of the transcription factor TFIIH. FIG. 1. Amino acids wereintroduced within the interaction motifs of the newly-identified planttransactivation domains to produce variant sequences.

We hypothesized that modifying the interaction motifs to contain the sixamino acid contact residues identified from the VP16 interaction motifof Subdomain II would produce modified interaction motifs of the plantactivators capable of interacting with a greater variety oftranscription factors, thereby resulting in higher levels of proteinexpression.

ERF2 Modifications.

The region from Asn53 to Ala85 in ERF2 aligned to the subdomain II ofVP16 transactivation domain (FIG. 2), and was identified as the planttransactivation domain sequence of ERF2. Modifications were introducedinto the region that was found to correspond to the interaction motif ofthe subdomain II of the VP16 transactivation domain; from Asp66 toAsp76. The amino acid residues that were different from the six contactresidues of the interaction motif of subdomain II of the VP 16transactivation domain were modified. These alterations resulted in anexemplary modified interaction motif of ERF2 (i.e., a variantinteraction motif sequence) that is similar to that of subdomain II ofthe VP16 transactivation domain. FIG. 3.

Modifications to PTI4, AtERF1, ORCA2, DREB1A, CBF1, and DOF1 InteractionMotifs.

Modifications were introduced into the PTI4, AtERF1, ORCA2, DREB1A, CBF1and DOF1 interaction motifs that are similar to those introduced intoERF2. The amino acid residues of the native sequences that weredifferent from the six direct contact residues of the interaction motifof subdomain II of the VP16 transactivation domain were modified,thereby producing exemplary variant interaction motifs. These changeswere introduced to make the plant variant interaction motifs similar(e.g., functionally similar) to that of subdomain II of the VP16transactivation domain. Exemplary sequences of these variant or modifiedinteraction motifs, as compared to the native interaction motif sequenceof subdomain II of the VP16 transactivation domain, are listed in FIGS.4-9.

Example 3 Testing the Interaction Motifs of the Plant Activation Domainsin a Saccharomyces cerevisiae Reporter Strain

Saccharomyces cerevisiae Reporter Strain.

A Saccharomyces cerevisiae reporter strain was produced to test plantactivation domains comprising either native or variant interactionmotifs. A three-step cloning procedure resulted in the construction ofthe yeast integration vector, pHO-zBG-MEL1 (FIG. 10).

First, two separate fragments of the yeast SSA reporter vector, pNorMEL1(Doyon et al. (2008) Nat. Biotechnol. 26(6):702-8), were amplified viaPCR. The first fragment contained a yeast KanMX expression cassette, andwas amplified from pNorMEL1 using the primers, KanMX-For (SEQ ID NO: 59)and KanMX-Rev (SEQ ID NO: 60), which add 5′ SpeI, BamHI, NheI, Pad,BglII, KpnI, and 3′ EcoRI restriction sites, respectively. The secondfragment contained outward-facing homology arms to the yeast HO locus(Voth et al. (2001) Nucleic Acids Res. 29(12):E59-9) separated by abacterial origin of replication, and was amplified using primers, HO-For(SEQ ID NO: 61) and HO-Rev (SEQ ID NO: 62). The two fragments weredigested with EcoRI/SpeI and ligated to generate a KanMX selectableHO-targeting vector.

Next, the MEL1 expression cassette from pMEL1α2 (Melcher et al. (2000)Gene 247(1-2):53-61) was amplified with MEL1-For (SEQ ID NO: 63) andMEL1-Rev (SEQ ID NO: 64) primers, and cloned into the KpnI site of theKanMX selectable HO-targeting vector.

Finally, a Zinc Finger Protein (ZFP)-binding site (referred to as “HAS”for High Affinity Site) was synthesized de novo by an external vendor(DNA2.0, Menlo Park, Calif.). This site contained binding sites forZinc-Finger Proteins (ZFPs) targeting the human CCR5 gene (Perez et al.(2008) Nat. Biotechnol. 26:808-16). The HAS fragment was PCR-amplifiedusing the primers, HAS-For-F1 (SEQ ID NO: 65) and HAS-For-R1 (SEQ ID NO:66), and cloned into the BamHI-Pad sites of the KanMX HO MEL1 vector,located upstream of the MEL1 reporter gene. This final vector wasdesignated, pHO-zBG-MEL1 (FIG. 10).

pHO-zBG-MEL1 was linearized with NotI to expose the flanking homologyarms for targeting to the yeast HO locus and transformed into S.cerevisiae strain, BY4741 MATα (Invitrogen, Carlsbad, Calif.), using themanufacturer's suggested protocol. Briefly, 3 mL of a log phase BY4741culture was pelleted, and washed in TEL buffer (10 mM Tris HCL pH 8.0, 1mM EDTA, 100 mM Lithium Acetate). The yeast cell pellet was resuspendedin 360 μL yeast transformation solution (33.3% PEG-3350 (Sigma-Aldrich,St. Louis, Mo.), 0.1 M Lithium Acetate (Sigma-Aldrich), and 0.2 mg/mLSalmon Sperm DNA (Stratagene, La Jolla, Calif.) in 1×TE) containing 3 μgof linearized pHO-zBG-MEL1, and heat-shocked for 40 minutes at 42° C.Yeast cells were pelleted, washed, and grown in rich medium for 2 hoursprior to selection on YPD plates containing 1 mg/L Geneticin® (LifeTechnologies, Carlsbad, Calif.). Resistant clones were re-streaked onYPD+Geneticin® plates, and used for subsequent transformations.

Yeast ZFP-Transcription Activator Expression Cassette Construction.

DNA constructs containing an in-frame CCR5 Zinc Finger Binding Protein(CCR5-ZFP)—plant transactivation interaction motif were constructed. Thenative and variant plant transactivation interaction motifs described inSEQ ID NOs: 80-93 were mobilized as a BamHI/HindIII restriction enzymefragment and cloned directly downstream of sequences encoding theCCR5-ZFP domains (Perez et al. (2008), supra). The resultingZFP-transcription activator expression cassette utilized a GAL1,10promoter (West et al. (1987) Genes Dev. 1:1118-31) and CYC1 terminator(Osborne et al. (1988) Genes Dev. 2:766-72), and was based on the yeastpRS315 series vector. The resulting vectors contained native and variantplant transactivation interaction motifs as in-frame fusions with theCCR5-ZFP.

In addition, several controls were included. An empty vector control andtwo different VP-16 transcription activator expression cassettes, SGMOVP16-CCR5 (SEQ ID NO: 79) and VP16v2 CCR5-CCR5, were used in the study.Both of the VP-16 transcription activator expression cassettes weredriven by the GAL 1,10 promoter, and terminated by the CYC1 terminator.The empty vector control contained only the CCR5-ZFP domains, and didnot contain a transactivation interaction motif.

Yeast Activity Assay.

Overnight cultures of the BY4741 reporter line strain were grown inYPD+Geneticin®, and 1 μg of vector containing a ZFP-transcriptionactivator expression cassette was delivered using a standard yeasttransformation protocol in a 96-well format. All transformations wereduplicated. Transformed yeast cells were recovered in Synthetic Dextrosemedium lacking leucine (SD-Leu) to select for the vector containing theZFP-transcription activator expression cassette. After 72 hours, theyeast cells were enriched by a 1:10 dilution of the transformants inSD-Leu and grown a further 24 hours. Next, the yeast cells were diluted1:10 into synthetic raffinose medium lacking leucine (SR-Leu) tode-repress the GAL1,10 promoter. 24 hours later, yeast cells werepelleted, and resuspended in synthetic galactose medium lacking leucine(SG-Leu). At time points of 0, 3, and 6 hours post-galactose induction,110 μL of yeast cells were harvested for a MEL1 assay.

In the MEL1 assay, 100 μL of the 110 μL of yeast cells were diluted in100 μL of water and the optical density at 600 nm (OD₆₀₀) was measuredusing a spectrophotometer. The remaining 10 μL of yeast cells wereincubated in 90 μL MEL1 buffer (77 mM Na₂HPO₄, 61 mM Citric Acid, 2mg/mL PNPG (Sigma-Aldrich)) for 1 hour at 30° C. The reaction wasstopped by the addition of 100 μL 1M Na₂CO₃. MEL1 activity was assessedat OD₄₀₅, and mU were calculated using a formula based on the ratio ofthe OD₄₀₅ and OD₆₀₀ measurements (Doyon et al. (2008) Nat. Biotechnol.26(6):702-8).

The expression level of the Mel1 reporter gene that resulted fromactivation by the different plant transactivation interaction motifs isshown in FIG. 11. The expression of the MEL1 protein that resulted fromthese different plant transactivation interaction motifs was compared toan empty vector control and the activation of Mel1 from subdomain II ofthe VP 16 transactivation domain (SEQ ID NO: 1) (VP16(v2)-CCR5) and SGMOVP16 (SGMO VP16-CCR5).

The modified ERF2 (v2) plant transactivation interaction motif producedunexpectedly high levels of expression, as compared to the VP16 control.In addition, expression of Mel1 with this variant plant transactivationinteraction motif resulted in an increase over the native version of theERF2 (v3) plant transactivation interaction motif.

The modified PTI4 (v2) plant transactivation interaction motif expressedMEL1 protein at levels similar to the VP16 transactivation domaincontrol. However, the modifications introduced into the PTI4 interactionmotif resulted in significantly higher levels of Mel1 expression, ascompared to the native PTI4 (v3) plant transactivation interactionmotif.

The AtERF1 (v3), AtERF1 (v2), ORCA2(v3), ORCA2(v2), DOF1 (v3), DOF1(v2), DREB1A(v3), DREB1A(v2), CBF1(v3) and CBF1(v2) planttransactivation interaction motifs did not result in high levels ofexpression of Mel1 in the yeast assay, as compared to the VP16 controls.However, the AtERF1, DREB1A, and CBF1 plant transactivation interactionmotifs did drive expression of Mel1 in yeast. Only the ORCA2 (v3) andDOF1 (v2) plant transactivation interaction motifs did not result in anyexpression of Mel1 in the yeast assay.

The levels of MEL1 produced by the plant transactivation domain for themodified variant (v2) plant transactivation interaction motifs weregenerally higher as compared to the native (v3) plant transactivationinteraction motifs in the Mel1 yeast assay. The only modified planttransactivation interaction motif which did not drive expression of Mel1in the yeast assay was the DOF1 (v2) interaction motif. This planttransactivation interaction motif did not produce any MEL1 expression inthe yeast assay.

Example 4 Function of Interaction Motifs of the Plant Activation Domainsin Tobacco Containing a Reporter Construct Comprising a Zinc Finger DNABinding Domain

Reporter Construct pDAB9897.

Eight tandem repeats of the Z6 DNA binding domain polynucleotidesequence (SEQ ID NO: 67; Yokoi et al. (2007) Mol. Ther. 15(11):1917-23)were synthesized de novo (IDT, Coralsville, Iowa) with SacII sites addedto the 5′ and 3′ ends to facilitate cloning. The entire 8X-Z6 bindingdomain (SEQ ID NO: 68) was subsequently cloned into a pre-existingGateway® Entry vector containing desired plant expression elements. The8X-Z6 binding sites were mobilized on a SacII fragment, and clonedimmediately upstream of the Arabidopsis thaliana actin-2 promoter(AtAct2 promoter v2; An et al. (1996) Plant J. 10:107-21) using a uniqueSacII site found in the backbone vector. Subsequently, the gus gene(GUS; Jefferson (1989) Nature 342:837-8) was cloned into this vectorunder direct control of the A. thaliana actin-2 promoter using uniqueNcoI/SacI sites, with the ATG codon of the NcoI site forming theinitiation codon. An Atu ORF23 3′UTR (Agrobacterium tumefaciens openreading frame-23, 3′ untranslated region; European Patent ApplicationNo. EP 222493 A1) was used to terminate transcription.

The final transformation vector, pDAB9897 (FIG. 12), was the result of aGateway® ligation with a destination vector containing an A. thalianaubiquitin-10 promoter (At Ubi10 Promoter v2 (Callis et al. (1990) J.Biol. Chem. 265:12486-93)) phosphinothricin acetyl transferase gene (patv3 (Wohlleben et al. (1988) Gene 70:25-37)): A. tumefaciens open readingframe-1,3′ untranslated region (AtuORF1 3′UTR v3 (Huang et al. (1990) J.Bacteriol. 172:1814-22) selectable marker cassette for plant selection.The final transformation vector was confirmed via sequencing, andtransformed into A. tumefaciens strain, LBA4404 (Invitrogen, Carlsbad,Calif.).

Agrobacterium-Mediated Transformation of Tobacco with pDAB9897.

To make transgenic reporter plant events, leaf discs (1 cm²) cut fromPetit Havana tobacco plants were incubated in an overnight culture of A.tumefaciens strain LBA4404 harboring plasmid pDAB9897, grown toOD₆₀₀˜1.2 nm, blotted dry on sterile filter paper, and then placed ontoMS medium (Phytotechnology Labs, Shawnee Mission, Kans.) and 30 g/Lsucrose with the addition of 1 mg/L indoleacetic acid and 1 mg/Lbenzyamino purine in 60×20 mm dishes (5 discs per dish) sealed withNescoflim® (Karlan Research Products Corporation, Cottonwood, Ariz.).Following 48 hours of co-cultivation, leaf discs were transferred to thesame medium with 250 mg/L cephotaxime and 5 mg/L BASTA°. After 3-4weeks, plantlets were transferred to MS medium with 250 mg/L cephotaximeand 10 mg/L BASTA® in PhytaTrays™ for an additional 2-3 weeks prior toleaf harvest and molecular analysis.

Copy Number and PTU Analysis of Reporter Events.

PCR DNA Isolation. Transgenic tobacco plant tissue was harvested fromnewly-grown plantlets and lyophilized (Labconco, Kansas City, Mo.) forat least 2 days in 96-well collection plates (Qiagen, Valencia, Calif.).DNA was then isolated using the DNEasy™ 96 well extraction kit (Qiagen),according to the manufacturer's instructions. A Model 2-96A Kleco™tissue pulverizer (Garcia Manufacturing, Visalia, Calif.) was used fortissue disruption.

Southern DNA Isolation. Transgenic tobacco plant tissue was harvestedfrom newly-grown plantlets and lyophilized (Labconco, Kansas City, Mo.)for at least 2 days in 2 mL conical tubes (Eppendorf). DNA was thenisolated using the DNEasy™ Plant Mini extraction kit (Qiagen), accordingto the manufacturer's instructions. A Model 2-96A Kleco™ tissuepulverizer (Garcia Manufacturing) was used for tissue disruption.

DNA Quantification. Resulting genomic DNA was quantified using aQuant-iT™ PicoGreen® DNA assay kit (Molecular Probes, Invitrogen,Carlsbad, Calif.). Five pre-quantified DNA standards ranging from 20ng/μL to 1.25 ng/μL (serially diluted) were used for standard curvegeneration. Samples were first diluted with 1:10 or 1:20 dilutions to bewithin the linear range of the assay, and concentrations of genomic DNAwere determined according to the manufacturer's protocol. Fluorescencewas then recorded using a Synergy2™ plate reader (Biotek, Winooski,Vt.). Genomic DNA concentration was estimated from a standard curvecalculated from background fluorescence corrections. Using TE or water,DNA was then diluted to a common concentration of 10 ng/μL for PCR usinga Biorobot3000™-automated liquid handler (Qiagen). DNA for Southernanalysis was left undiluted.

Copy Number Estimation. Putative transgenic events were analyzed forintegration complexity using a multiplexed DNA hydrolysis probe assayanalogous to the TaqMan® assay (Applied Biosystems, Carlsbad, Calif.).The copy number of the transgene insert was estimated usingsequence-specific primers and probes for both the pat transgene and anendogenous tobacco reference gene, pal (phenylalanine ammonium lyase;GenBank Accession No. AB008199). Assays for both genes were designedusing LightCycler® Probe Design Software 2.0 (Roche Applied Science,Indianapolis, Ind.). Real time PCR for both genes was evaluated usingthe LightCycler®480 system (Roche Applied Science).

For amplification, LightCycler®480 Probes Master mix (Roche AppliedScience) was prepared at 1× final concentration in a 10 pit volumemultiplex reaction containing 0.4 μM of each primer and 0.2 μM of eachprobe. Table 1. A two-step amplification reaction was performed with anextension at 58° C. for 38 seconds with fluorescence acquisition. Allsamples were run in triplicate, and the averaged Ct values were used foranalysis of each sample. Analysis of real time PCR data was performedusing LightCycler® software (Roche Applied Science) via the relativequant module, and is based on the ΔΔCt method. A sample of gDNA from asingle copy calibrator was included to normalize results. The singlecopy calibrator event was previously identified by Southern analysis,and was confirmed to have a single insert of the pat gene.

TABLE 1 Sequences of the primers and probes used in both the patand pal hydrolysis probe (HP) assays. The fluorescentepitope of each probe was different, which allowed the assays to be run simultaneously as a multiplexed reaction. PrimerNucleotide Sequence (5′-3′) Type TQPATS ACAAGAGTGGATTGATGATCTAGAGAGGTPrimer (SEQ ID NO: 69) TQPATA CTTTGATGCCTATGTGACACGTAAACAGT Primer(SEQ ID NO: 70) TQPATFQ CY5-GGTGTTGTGGCTGGTATTGCTTACGCTGG-BHQ2 Cy5(SEQ ID NO: 71) Probe TQPALS TACTATGACTTGATGTTGTGTGGTGACTGA Primer(SEQ ID NO: 72) TQPALA GAGCGGTCTAAATTCCGACCCTTATTTC Primer(SEQ ID NO: 73) TQPALFQ 6FAM-AAACGATGGCAGGAGTGCCCTTTTTCTATCAAT-BHQ1 6FAM(SEQ ID NO: 74) Probe

PTU PCR. Low copy events were subsequently screened by PCR for intactplant transcriptional units (PTU). Using the Blu636 (SEQ ID NO: 75) andBlu637 (SEQ ID NO: 76) primers, correct amplification resulted in a 3.7kb product consisting of the Z6-Act2/GUS/AtORF23 PTU (3,771 bp).Phusion® GC Master Mix (New England Biolabs, Beverley, Mass.) was usedwith the following reaction conditions: 98° C. for 30 seconds, followedby 30 cycles of 98° C. for 10 seconds, 67° C. for 30 seconds, 72° C. for2 minutes, and a final extension of 72° C. for 10 minutes. In additionto the PTU PCR reaction detailed above, amplification of an endogenousgene, chs (chalcone synthase; Genbank Accession No. FJ969391.1), wasalso included to confirm the quality of the genomic DNA templates. 3′CHSForward (SEQ ID NO: 77) and 3′CHS Reverse (SEQ ID NO: 78) primers wereincluded in the reaction, which produced a 350 bp amplification product.20 μL reactions were used, with a final concentration of 0.5 μM for thetransgene primers and 0.2 μM for the endogenous reference gene.

Southern Analysis. For each sample, 5 μg genomic DNA was thoroughlydigested with the restriction enzyme, AseI (New England Biolabs), byincubation at 37° C. overnight. The digested DNA was concentrated byprecipitation with Quick Precip™ Solution (Edge Biosystems,Gaithersburg, Md.), according to the manufacturer's suggested protocol.The genomic DNA was then resuspended in 25 μL water at 65° C. for 1hour. Resuspended samples were loaded onto a 0.8% agarose gel preparedin 1×TAE buffer, and electrophoresed overnight at 1.1 V/cm in 1×TAE. Thegel was immersed in denaturation buffer (0.2 M NaOH/0.6 M NaCl) for 30minutes, followed by immersion in neutralization buffer (0.5 M Tris-HCl(pH 7.5)/1.5 M NaCl) for 30 minutes.

Transfer of DNA fragments to nylon membranes was performed by passivelywicking 20×SSC buffer overnight through the gel onto treatedImmobilon™-NY+ transfer membrane (Millipore, Billerica, Mass.) using achromatography paper wick and paper towels. Following transfer, themembrane was briefly washed with 2×SSC, cross-linked with theStratalinker® 1800 (Stratagene, LaJolla, Calif.), and vacuum baked at80° C. for 3 hours.

Blots were incubated with pre-hybridization solution (PerfectHyb™ plus,Sigma-Aldrich) for 1 hour at 65° C. in glass roller bottles using aRobbins Scientific Model 400 hybridization incubator (RobbinsScientific, Sunnyvale, Calif.). Probes were prepared from a PCR fragmentcontaining the entire coding sequence. The PCR amplicon was purifiedusing Qiaex II® gel extraction kit (Qiagen), and labeled with [α³²P]dCTPvia the Random RT Prime-iT® labeling kit (Stratagene, La Jolla, Calif.).Blots were hybridized overnight at 65° C. with a denatured probe addeddirectly to the pre-hybridization solution to approximately 2 millioncounts blot⁻¹ mL⁻¹. Following hybridization, blots were sequentiallywashed at 65° C. with 0.1×SSC/0.1% SDS for 40 minutes. Finally, theblots were exposed to storage phosphor imaging screens, and imaged usinga Molecular Dynamics™ Storm™ 860 imaging system. True single copyintegration events were confirmed by the identification of a singlehybridizing band.

Generation of Homozygous T₂ Reporter Plants.

A total of 51 BASTA®-resistant plants were generated, of which 24 werefound to be low-complexity (1-2 copies of pat) based on hydrolysis probeanalysis of copy number. Of these low-complexity events, 18 displayed anintact PTU, as determined by PCR analysis. Following Southern blotanalysis, two single-copy, intact PTU events were selected and grown tomaturity in the greenhouse, where they were allowed to self-pollinate.T₁ seed was then collected, surface-sterilized (for 3 min in 20% bleach,followed by two sterile, distilled water rinses), and germinated on MSmedium (Phytotechnology Labs, Shawnee Mission, Kans.) and 30 g/L sucrosein PhytaTrays™ (Sigma, St. Louis, Mo.). Following zygosity screening viapat copy number analysis, homozygous T₁ plants were selected, grown tomaturity in the greenhouse, and allowed to self-pollinate. T₂ seed wasthen collected, surface-sterilized, and germinated as describedpreviously, and used to generate reporter plants for planttransactivation testing.

Plant ZFP-Plant Transcription Activator Expression Constructs.

Plant ZFP-transcription activator constructs containing a variant ornative plant transactivation interaction motif were constructed. Planttransactivation interaction motifs (both native and modified variants)flanked by the restriction enzyme sites BamHI/SacI for cloning weresynthesized de novo (DNA2.0, Menlo Park, Calif.). The planttransactivation interaction motifs were mobilized on a BamHI/SacIfragment, and cloned immediately downstream of the Z6 Zinc Finger DNAbinding domain (Yokoi et al. (2007), supra) using unique BamHI/SacIsites found in an existing Gateway® Entry backbone vector. Uponcompletion of this step, the ZFP-transcription activator constructs(containing a Z6 DNA Zinc Finger Protein binding domain fused to theplant transactivation interaction motif) were placed under the controlof the constitutive Cassava Vein Mosaic Virus promoter (CsVMV promoterv2; Verdaguer et al. (1996) Plant Mol. Biol. 31:1129-39), and terminatedwith the ORF23 3′UTR from A. tumefaciens. Final transformation vectors(Table 2), resulted from a Gateway®-mediated ligation (Invitrogen,Carlsbad, Calif.) with a destination vector containing an A. thalianaubiquitin-3 promoter (At Ubi3 promoter v2; Callis et al. (1995)Genetics, 139(2):921-39))/hygromycin phosphotransferase II (HPTII v1;Gritz et al. (1983) Gene 25(2-3):179-88)/A. tumefaciens open readingframe-24, 3′ untranslated region (Atu ORF24 3′UTR v2) cassette used forplant selection.

TABLE 2 Plant ZFP-transcription activator constructs tested in tobacco.The sequence identifier provides the DNA sequence of the planttransactivation interaction motif that was fused to the Z6 Zinc Fingerbinding Protein and expressed in the binary vector. PlantTransactivation Interaction Construct Number and Plant TransactivationInteraction Motif Sequence Motif Native Modified Pti4 pDAB107881 FIG. 13pDAB106273 FIG. 22 SEQ ID NO: 81 SEQ ID NO: 88 AtERF1 pDAB107882 FIG. 14pDAB106274 FIG. 23 SEQ ID NO: 82 SEQ ID NO: 89 ORCA2 pDAB107883 FIG. 15pDAB106275 FIG. 24 SEQ ID NO: 83 SEQ ID NO: 90 Dreb1a pDAB107884 FIG. 16pDAB106276 FIG. 25 SEQ ID NO: 84 SEQ ID NO: 91 Dof1 pDAB107885 FIG. 17pDAB106277 FIG. 26 SEQ ID NO: 86 SEQ ID NO: 93 ERF2 pDAB107886 FIG. 18pDAB106278 FIG. 27 SEQ ID NO: 80 SEQ ID NO: 87 Cbf1 pDAB107887 FIG. 19pDAB106279 FIG. 28 SEQ ID NO: 85 SEQ ID NO: 92 VP16 pDAB106272 FIG. 20 —— SEQ ID NO: 79 Empty Vector - pDAB106238 FIG. 21 — — Zinc Finger Only(no transactivation interaction motif)

The final binary vector was confirmed via DNA sequencing, andtransformed into A. tumefaciens strain, LBA4404 (Invitrogen). Inaddition, a control vector pDAB106238 (FIG. 21), which contains the zincfinder binding domain and does not include an activator composed of thetransactivation interaction motif, was included. In this construct, thezinc finger binding domain was placed under the control of theconstitutive A. tumefaciens MAS promoter (AtuMas promoter v4; U.S. Pat.Nos. 5,001,060; 5,573,932 and 5,290,924), and terminated with the ORF233′UTR from A. tumefaciens. In addition, the vector contains the A.thaliana ubiquitin-3 promoter/hygromycin phosphotransferase II/A.tumefaciens open reading frame-24, 3′ untranslated region cassette usedfor plant selection.

To produce plant events containing the plant ZFP-transcription activatorconstructs, leaf discs (1 cm²) cut from T₂ reporter tobacco plants wereincubated in an overnight culture of A. tumefaciens strain, LBA4404(Invitrogen, Carlsbad, Calif.), harboring one of the 16 plasmids listedin Table 2, grown to OD₆₀₀˜1.2 nm, blotted dry on sterile filter paper,and then placed onto MS medium (Phytotechnology Labs, Shawnee Mission,Kans.) and 30 g/L sucrose with the addition of 1 mg/L indoleacetic acidand 1 mg/L benzyamino purine in 60×20 mm dishes (5 discs per dish)sealed with Nescofilm® (Karlan Research Products Corporation,Cottonwood, Ariz.). Following 48 hours of co-cultivation, leaf discswere transferred to the same medium with 250 mg/L cephotaxime and 10mg/L hygromycin. After 3-4 weeks, plantlets were transferred to MSmedium with 250 mg/L cephotaxime and 10 mg/L hygromycin in PhytaTrays™for an additional 2-3 weeks, followed by leaf harvest and gus expressionanalysis. A total of 20-30 plant events were generated for each of the16 plant transcription activator constructs.

gus Expression Analysis.

mRNA Isolation. Transgenic tobacco plant tissue was harvested from newlygrowing plantlets and flash frozen on dry ice in 96-well collectionplates (Qiagen). RNA was then isolated using the RNEasy® 96-wellextraction kit (Qiagen), according to the manufacturer's instructions. AModel 2-96A Kleco™ tissue pulverizer (Garcia Manufacturing) was used fortissue disruption.

RNA Quantification. Resulting mRNA was quantified using a NanoDrop™ 8000spectrophotometer (Thermo Scientific, Wilmington, Del.). Each well wasblanked with 4 μL RNase-free water prior to loading and quantify 4 μL ofundiluted samples. mRNA concentration was estimated from NanoDrop™ 8000software, using the standard RNA nucleic acid measurement method. mRNAwas hand-diluted with RNase free water to ˜83 ng/μL.

cDNA Preparation. cDNA was prepared from diluted mRNA using theQuantitect® RT kit (Qiagen, Carlsbad, Calif.), following themanufacturer's instructions. 1 μg total mRNA was used in each reaction.Upon completion, cDNA was stored at −20° C. until analysis wascompleted.

RT-PCR. Events selected on hygromycin were analyzed for gus genetranscript levels using two DNA hydrolysis probe assays, both of whichare analogous to TaqMan® assays. Steady state levels of gus mRNA foreach individual event were estimated using sequence-specific primers andprobe. The mRNA was normalized using the steady state level of mRNA foran endogenous tobacco reference gene, BYEEF (Genbank Accession No.GI:927382). Assays for both genes were designed using LightCycler® ProbeDesign Software 2.0 (Roche Applied Science). Real time PCR for bothgenes was evaluated using the LightCycler® 480 system. For gusamplification, LightCycler® 480 Probes Master mix was prepared at 1×final concentration in a 10 μL volume multiplex reaction containing 0.4μM of each primer and 0.2 μM probe. Table 3.

A two-step amplification reaction was performed with an extension at 56°C. for 40 seconds with fluorescence acquisition. All samples were runundiluted in triplicate, and the averaged Ct values were used foranalysis of each sample. For BYEEF amplification, LightCycler® 480Probes Master mix was prepared at 1× final concentration in a 10 μLvolume multiplex reaction containing 0.25 μM of each primer (Table 3)and 0.1 μM UPL 119 probe (Roche Applied Science). A two-stepamplification reaction was performed with an extension at 58° C. for 25seconds with fluorescence acquisition. All samples were run diluted 1:10in triplicate, and the averaged Ct values were used for analysis of eachsample. Analysis of real time PCR data was performed using LightCycler®software using the relative quant module, and is based on the ΔΔCtmethod. Relative expression levels amongst the different planttranscription activator treatments were then compared. FIG. 29.

TABLE 3 Sequences of the primers and probes used in boththe gus and BYEEF hydrolysis probe (HP) assays. PrimerNucleotide Sequence (5′-3′) Type TQGUSS AGACAGAGTGTGATATCTACCC Primer(SEQ ID NO: 75) TQGUSA CCATCAGCACGTTATCGAAT Primer (SEQ ID NO: 76)TQGUSFQ 6FAM-CACAAACCGTTCTACTTTACTGGCTT- 6FAM BHQ1 Probe (SEQ ID NO: 77)BYEEFU119F AGGCTCCCACTTCAGGATG Primer (SEQ ID NO: 78) BYEEFU119RCACGACCAACAGGGACAGTA Primer (SEQ ID NO: 79)

Results.

FIG. 29 shows the resulting ratio of gus transcript levels for thedifferent plant transactivation interaction motifs, as normalized byBYEEF endogenous gene expression levels. The activation of the gus genefrom the different plant transactivation interaction motifs was comparedto an empty vector control, and the interaction motif of subdomain II ofthe VP16 transactivation domain. Several of the plant transactivationinteraction motifs showed unexpectedly high levels of expression ascompared to subdomain II of the VP16 transactivator. For instance, thePTI4, DREB1A, ERF2, and CBF1 plant transactivation interaction motifsexpressed more gus mRNA than subdomain II of the VP16 transcriptionactivation domain.

The levels of mRNA produced by the plant transactivation interactionmotif for the modified variant (v2) as compared to the native version(v3) varied amongst the plant transactivation interaction motifs. Themodified version of the ERF2 plant transactivation interaction motifproduced significantly more gus mRNA than the ERF2 native sequenceinteraction motif Likewise, the modified CBF1 plant transactivationinteraction motif produced more mRNA than the CBF1 native sequenceinteraction motif Conversely, the modifications introduced within thePTI4 and DREB1A transactivation interaction motif resulted in theproduction of lower gus mRNA levels, as compared to the native versionsof PTI4 and DREB1A plant transactivation interaction motifs.

Example 5 Interaction Motif Function in Tobacco Containing a GAL4Reporter Construct Tobacco Line Containing a Reporter ConstructComprised of a GAL4 Binding Domain

The reporter construct, pGalGUS, is built using the strategy describedbelow. Six tandem repeats of the yeast GAL4 binding sequence and 23 bpspacer regions (as described in Baleja et al. (1997) J. Biomol. NMR10:397-401) are synthesized de novo (IDT) with added SacII sites tofacilitate cloning. The 6X Gal4 binding sites are mobilized on a SacIIfragment, and are used to replace the Z6 binding sites from apre-existing entry vector that is also digested with SacII. This cloningstep places the GAL4 binding sites immediately upstream of theArabidopsis Actin 2 promoter, which drives expression of the gus gene.The final transformation vector, pGalGUS (FIG. 30), results from aGateway® Transformation reaction with a destination vector containing anArabidopsis Ubiquitin10 promoter-pat gene expression cassette, which isused for plant selection. The final transformation vector is confirmedvia sequencing, and transformed into A. tumefaciens strain, LBA4404(Invitrogen).

Agrobacterium-Mediated Transformation of Tobacco with pGALGUS.

Transgenic reporter plants are made using the protocol described above.See “Agrobacterium-mediated transformation of tobacco with pDAB9897.”

Copy Number and PTU Analysis of Reporter Events.

Low-complexity copy number, BASTA®-resistant transgenic plants aregenerated and identified based on TaqMan® copy number analysis. Of thelow-complexity events, a subset displays an intact PTU, as determined byPCR analysis. These events are further analyzed via Southern blotanalysis. Following Southern blot analysis, at least one single-copy,intact PTU event is selected and grown to maturity in a greenhouse, andis allowed to self-pollinate. T₁ seed is collected, surface sterilized,and germinated. Following zygosity screening via pat copy numberanalysis, homozygous T₁ plants are selected, grown to maturity in thegreenhouse, and allowed to self-pollinate. T₂ seed is then collected,surface sterilized, and germinated (as described previously), and isused to generate reporter plants for activator testing.

Plant GAL4-Transcriptional Activator Expression Constructs.

Plant GAL4-transcription activator constructs containing a variant ornative plant transactivation interaction motif are constructed. Theplant ZFP-transcription activator expression constructs described inExample 4 (“Plant ZFP-Plant Transcription Activator ExpressionConstructs”) are modified by inserting a GAL4 binding proteinpolynucleotide sequence in place of the Zinc Finger Binding Proteinpolynucleotide sequence. The hemicot plant-optimized GAL4 DNA bindingdomain polynucleotide sequence (Keegan et al. (1986) Science231(4739):699-704) is inserted in place of the Zinc Finger BindingProtein polynucleotide sequence as an NcoI/BamHI fragment. Uponcompletion of this step, the GAL4-transcription activator construct isplaced under the control of the constitutive Cassava Vein Mosaic Viruspromoter, and terminated with the ORF23 3′UTR from A. tumefaciens. Finalbinary transformation vectors are completed, resulting from a Gateway®transformation with a destination vector containing an ArabidopsisUbiquitin3-HptII cassette for plant selection. The final transformationvector is confirmed via sequencing, and transformed into A. tumefaciensstrain, LBA4404 (Invitrogen).

To produce plant events containing a plant GAL4-transcription activatorconstruct, the transient transformation protocol described in Example 4is used. A total of 20-30 plant events are generated for each of the 16GAL4-transcription activator constructs.

gus Expression Analysis.

Events selected on hygromycin are analyzed for gus gene transcriptlevels using two DNA hydrolysis probe assays. Steady state levels of gusmRNA for each individual event are estimated using sequence specificprimers and a probe. The mRNA for each event is normalized using thesteady state level of mRNA for an endogenous tobacco reference gene,e.g., BYEEF. Assays for both genes are designed using the protocoldescribed in Example 4. Analysis of real time PCR data is performedusing LightCycler® software using the relative quant module, and isbased on the ΔΔCt method. Relative expression levels for the differentactivator constructs are compared. The results indicate that planttransactivation interaction motifs and engineered variants of theseplant transactivation interaction motifs can be used as transcriptionalactivators, and can be fused with a GAL4 binding protein fortranscriptional activation of a gene.

Example 6 Interaction Motif Function in Tobacco Containing a TALReporter Construct

Tobacco Line Containing a Reporter Construct Comprised of a TAL BindingDomain.

The reporter construct, pTalGUS, is built using the strategy describedbelow. Eight tandem repeats sequences (TATATAAACCTNNCCCTCT (SEQ ID NO:99)) taken from the consensus binding sequence of AVRBS3-induciblegenes, and termed the UPA DNA binding domain (Kay et al. (2009) Plant J.59(6):859-71), are synthesized de novo (IDT) with added SacII sites tofacilitate cloning.

The 8×UPA binding sites are mobilized on a SacII fragment, and are usedto replace the Z6 binding sites from a pre-existing entry vector whichis also digested with SacII. This cloning step places the UPA bindingsites immediately upstream of the Arabidopsis Actin 2 promoter, whichdrives expression of the gus gene. The final transformation vector,pTalGUS (FIG. 31), results from a Gateway® Transformation reaction witha destination vector containing an A. thaliana Ubiquitin10 promoter/patgene expression cassette, which is used for plant selection. The finaltransformation vector is confirmed via sequencing and transformed intoA. tumefaciens strain, LBA4404 (Invitrogen).

Agrobacterium-Mediated Transformation of Tobacco with pTALGUS.

Transgenic reporter plants are made using the protocol described above.See “Agrobacterium-mediated transformation of tobacco with pDAB9897.”

Copy Number and PTU Analysis of Reporter Events.

Low-complexity copy number, BASTA®-resistant, transgenic plants aregenerated and identified utilizing a TaqMan® copy number analysis. Ofthe low-complexity events, a subset displays an intact PTU, asdetermined by PCR analysis. These events are further analyzed viaSouthern blot analysis. Following Southern blot analysis, at least 1single-copy, intact PTU event is selected and grown to maturity in agreenhouse, and is allowed to self-pollinate. T₁ seed is collected,surface sterilized, and germinated. Following zygosity screening via patcopy number analysis, homozygous T₁ plants are selected, grown tomaturity in the greenhouse, and allowed to self-pollinate. T₂ seed isthen collected, surface sterilized, and germinated (as describedpreviously), and is used to generate reporter plants for activatortesting.

Plant TAL-Transcriptional Activator Expression Constructs.

Plant TAL-transcription activator constructs containing a variant ornative plant transactivation interaction motif are constructed. Theplant ZFP-transcription activator expression constructs described inExample 4 are modified by inserting a TAL binding protein polynucleotidesequence in place of the Zinc Finger Binding Protein polynucleotidesequence. The 17.5 TAL repeats which are needed for DNA binding aresynthesized de novo, and fused to a Zea mays Opaque-2 nuclearlocalization sequence (Van Eenennaam et al. (2004) Metabolic Engineering6:101-8). The sequence of each domain utilizes different amino acids atthe variable residues (12 and 13 position) to dictate DNA binding, aspredicted for the UPA-box consensus sequence. Boch et al. (2009) Science326(5959):1509-12. The hemicot plant-optimized TAL DNA binding domainpolynucleotide sequence is inserted in place of the Zinc Finger BindingProtein polynucleotide sequence as an NcoI/BamHI fragment. Uponcompletion of this step, the TAL-transcription activator construct isplaced under the control of the constitutive Cassava Vein Mosaic Viruspromoter, and terminated with the ORF23 3′UTR from A. tumefaciens. Finaltransformation vectors are completed from a Gateway® transformation witha destination vector containing an Arabidopsis Ubiquitin 3-HptIIcassette for plant selection. The final transformation vector isconfirmed via sequencing and transformed into A. tumefaciens strain,LBA4404 (Invitrogen).

To produce plant events containing a plant TAL-transcription activatorconstruct, the transient transformation protocol described in Example 4is used. A total of 20-30 plant events are generated for each of the 16TAL-transcription activator constructs.

gus Expression Analysis.

Events selected on hygromycin are analyzed for gus gene transcriptlevels using two DNA hydrolysis probe assays. Steady state levels of gusmRNA for each individual event are estimated using sequence specificprimers and a probe. The mRNA is normalized using the steady state levelof mRNA for an endogenous tobacco reference gene, e.g., BYEEF. Assaysfor both genes are designed using the protocol described in Example 4.Analysis of real time PCR data is performed using LightCycler® softwareusing the relative quant module, and is based on the ΔΔCt method.Relative expression levels for the different activator constructs arecompared.

1. A synthetic transcriptional activator fusion protein comprising: atleast one DNA-binding polypeptide; and at least one transactivationdomain interaction motif polypeptide having at least 80% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 10-58.
 2. The synthetic transcriptional activator fusionprotein of claim 1, wherein the DNA-binding polypeptide is selected fromthe group consisting of a zinc finger DNA-binding domain; a consensusbinding sequence from a AVRBS3-inducible gene or synthetic bindingsequence engineered therefrom; GAL4; TAL; LexA; a Tet repressor; LacR;and a steroid hormone receptor.
 3. (canceled)
 4. The synthetictranscriptional activator fusion protein of claim 1, wherein thetranscriptional activator fusion protein comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 2-8 and SEQID NOs: 100-120.
 5. The synthetic transcriptional activator fusionprotein of claim 1, wherein the transactivation domain interaction motifpolypeptide is selected from the group consisting of SEQ ID NOs: 10-58.6.-11. (canceled)
 12. The synthetic transcriptional activator fusionprotein of claim 1, wherein the fusion protein increases the expressionof a nucleotide sequence of interest when the nucleotide sequence ofinterest is operably linked to a second nucleotide sequence, and whereinthe fusion protein specifically binds to the second nucleotide sequence.13.-16. (canceled)
 17. A nucleic acid encoding the synthetictranscriptional activator fusion protein of claim 1, the nucleic acidcomprising: at least one first polynucleotide sequence encoding aDNA-binding polypeptide; and at least one second polynucleotide sequenceencoding a transactivation domain interaction motif polypeptide havingat least 80% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 10-58, wherein the first and secondpolynucleotide sequences are expressed from the nucleic acid in frameand in a single transcript.
 18. (canceled)
 19. The nucleic acid of claim17, wherein the nucleic acid comprises at least one polynucleotidesequence encoding a transactivation domain interaction polypeptideselected from the group consisting of SEQ ID NOs: 10-58.
 20. The nucleicacid of claim 17, wherein the first and second polynucleotide sequencesare operably linked to a gene regulatory element.
 21. The nucleic acidof claim 17, wherein the first and second polynucleotide sequences areseparated by a third polynucleotide sequence.
 22. The nucleic acid ofclaim 17, wherein the nucleic acid is integrated into the genome of ahost cell.
 23. The nucleic acid of claim 22, wherein the host cell is aplant cell.
 24. (canceled)
 25. The nucleic acid of claim 17, wherein theDNA-binding polypeptide binds specifically to a sequence selected fromthe group consisting of SEQ ID NOs: 67, SEQ ID NO: 68, and SEQ ID NO:99.
 26. A vector comprising the nucleic acid of claim
 17. 27.-29.(canceled)
 30. The nucleic acid of claim 17, wherein the nucleic acidcomprises a nucleotide sequence having at least 80% identity to anucleotide sequence selected from the group consisting of SEQ ID NO:80-93. 31.-35. (canceled)
 36. The nucleic acid of claim 30, wherein thenucleic acid comprises a nucleotide sequence selected from the groupconsisting of: SEQ ID NO: 80-93; a nucleotide sequence that issubstantially identical to one of SEQ ID NOs: 80-93; the complement of apolynucleotide that is specifically hybridizable to at least one of SEQID NOs: 80-93; and the reverse complement of a polynucleotide that isspecifically hybridizable to at least one of SEQ ID NOs: 80-93. 37.(canceled)
 38. The nucleic acid of claim 36, wherein the nucleic acidcomprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 80-93.
 39. A cell comprising the nucleic acidof claim
 17. 40. The cell of claim 39, wherein the cell is a plant cellor a yeast cell. 41.-42. (canceled)
 43. A plant tissue, plant part,plant commodity product, or whole plant comprising the cell of claim 39.44. (canceled)
 45. A method for increasing the expression of anucleotide sequence of interest in a host cell, the method comprising:introducing the nucleic acid of claim 17 into a host cell comprising thenucleotide sequence of interest, wherein the nucleotide sequence ofinterest is operably linked to a second nucleotide sequence that bindsspecifically to the DNA-binding polypeptide, thereby increasing theexpression of the nucleotide sequence of interest in the host cell. 46.The method according to claim 45, wherein introducing the nucleic acidinto the host cell comprises introducing a vector comprising the nucleicacid into the host cell. 47.-49. (canceled)
 50. The method according toclaim 45, wherein introducing the nucleic acid into the host cellcomprises crossing a plant comprising the nucleic acid with a plantcomprising the host cell.
 51. The method according to claim 45, whereinthe method comprises introducing into the host cell a nucleic acidcomprising the nucleotide sequence of interest operably linked to thesecond nucleotide sequence that binds specifically to the DNA-bindingpolypeptide.
 52. The method according to claim 51, wherein introducingthe nucleic acid comprising the nucleotide sequence of interest into thehost cell comprises introducing a vector comprising the nucleotidesequence of interest into the host cell. 53-54. (canceled)
 55. Themethod according to claim 51, wherein introducing the nucleic acidcomprising the nucleotide sequence of interest into the host cellcomprises crossing a plant comprising the nucleotide sequence ofinterest with a plant comprising the host cell.
 56. (canceled)
 57. Anucleic acid encoding a synthetic transcriptional activator fusionprotein, the nucleic acid comprising: a first polynucleotide sequenceencoding a DNA-binding polypeptide; and a second polynucleotide sequenceencoding means for transactivation of plant gene expression, wherein thefirst and second polynucleotide sequences are expressed from the nucleicacid in frame and in a single transcript.
 58. A method for increasingthe expression of a nucleotide sequence of interest in a host cell, themethod comprising: introducing the nucleic acid of claim 57 into a hostcell comprising the nucleotide sequence of interest, wherein thenucleotide sequence of interest is operably linked to a secondnucleotide sequence that binds specifically to the DNA-bindingpolypeptide, thereby increasing the expression of the nucleotidesequence of interest in the host cell.
 59. The method according to claim58, wherein the method comprises introducing into the host cell thenucleotide sequence of interest operably linked to the second nucleotidesequence that binds specifically to the DNA-binding polypeptide.
 60. Anucleic acid comprising a nucleotide sequence encoding a transactivationdomain interaction motif polypeptide selected from the group consistingof SEQ ID NOs: 121-127.
 61. The complement or reverse complement of anucleic acid that is specifically hybridizable to the nucleic acid ofclaim
 60. 62.-65. (canceled)
 66. A nucleic acid that is specificallyhybridizable to the nucleic acid of claim
 19. 67. The complement orreverse complement of the nucleic acid of claim 66.